Inhalable dry powder and aerosol formulations of jak inhibitors

ABSTRACT

wherein R1, R2, R3, R4, R5 and R6 are as defined herein, and salts thereof that are useful as JAK kinse inhibitors are described herein. Also provided are pharmaceutical compositions that include such a JAK inhibitor and a pharmaceutically acceptable carrier, adjuvant or vehicle, and methods of treating or lessening the severity of a disease or condition responsive to the inhibition of a Janus kinase activity in a patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/902,499 filed on Jun. 16, 2020, which claims priority toInternational Application No. PCT/CN2019/091712 filed on Jun. 18, 2019,and U.S. Provisional Application No. 63/035,381 filed on Jun. 5, 2020,the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to inhalable dry powder and aerosol formulationsof Janus kinases, such as JAK1 and JAK2, as well as compositionscontaining these compounds, and methods of use including, but notlimited to, diagnosis or treatment of patients suffering from acondition responsive to the inhibition of a JAK kinase.

BACKGROUND OF INVENTION

Cytokine pathways mediate a broad range of biological functions,including many aspects of inflammation and immunity. Janus kinases(JAK), including JAK1, JAK2, JAK3 and TYK2, are cytoplasmic proteinkinases that associate with type I and type II cytokine receptors andregulate cytokine signal transduction. Cytokine engagement with cognatereceptors triggers activation of receptor associated JAKs and this leadsto JAK-mediated tyrosine phosphorylation of signal transducer andactivator of transcription (STAT) proteins and ultimatelytranscriptional activation of specific gene sets (Schindler et al.,2007, J. Biol. Chem. 282: 20059-63). JAK1, JAK2 and TYK2 exhibit broadpatterns of gene expression, while JAK3 expression is limited toleukocytes. Cytokine receptors are typically functional as heterodimers,and as a result, more than one type of JAK kinase is usually associatedwith cytokine receptor complexes. The specific JAKs associated withdifferent cytokine receptor complexes have been determined in many casesthrough genetic studies and corroborated by other experimental evidence.Exemplary therapeutic benefits of the inhibition of JAK enzymes arediscussed, for example, in International Application No. WO 2013/014567.

JAK1 was initially identified in a screen for novel kinases (Wilks A.F., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1603-1607). Genetic andbiochemical studies have shown that JAK1 is functionally and physicallyassociated with the type I interferon (e.g., IFNalpha), type IIinterferon (e.g., IFNgamma), and IL-2 and IL-6 cytokine receptorcomplexes (Kisseleva et al., 2002, Gene 285:1-24; Levy et al., 2005,Nat. Rev. Mol. Cell Biol. 3:651-662; O'Shea et al., 2002, Cell, 109(suppl.): S121-S131). JAK1 knockout mice die perinatally due to defectsin LIF receptor signaling (Kisseleva et al., 2002, Gene 285:1-24; O'Sheaet al., 2002, Cell, 109 (suppl.): S121-S131). Characterization oftissues derived from JAK1 knockout mice demonstrated critical roles forthis kinase in the IFN, IL-10, IL-2/IL-4 and IL-6 pathways. A humanizedmonoclonal antibody targeting the IL-6 pathway (Tocilizumab) wasapproved by the European Commission for the treatment ofmoderate-to-severe rheumatoid arthritis (Scheinecker et al., 2009, Nat.Rev. Drug Discov. 8:273-274).

CD4 T cells play an important role in asthma pathogenesis through theproduction of TH2 cytokines within the lung, including IL-4, IL-9 andIL-13 (Cohn et al., 2004, Annu. Rev. Immunol. 22:789-815). IL-4 andIL-13 induce increased mucus production, recruitment of eosinophils tothe lung, and increased production of IgE (Kasaian et al., 2008,Biochem. Pharmacol. 76(2): 147-155). IL-9 leads to mast cell activation,which exacerbates the asthma symptoms (Kearley et al., 2011, Am. J.Resp. Crit. Care Med., 183(7): 865-875). The IL-4Rα chain activates JAK1and binds to either IL-4 or IL-13 when combined with the common gammachain or the IL-13Rα1 chain respectively (Pernis et al., 2002, J. Clin.Invest. 109(10):1279-1283). The common gamma chain can also combine withIL-9Rα to bind to IL-9, and IL-9Rα activates JAK1 as well (Demoulin etal., 1996, Mol. Cell Biol. 16(9):4710-4716). While the common gammachain activates JAK3, it has been shown that JAK1 is dominant over JAK3,and inhibition of JAK1 is sufficient to inactivate signaling through thecommon gamma chain despite JAK3 activity (Haan et al., 2011, Chem. Biol.18(3):314-323). Inhibition of IL-4, IL-13 and IL-9 signaling by blockingthe JAK/STAT signaling pathway can alleviate asthmatic symptoms inpre-clinical lung inflammation models (Mathew et al., 2001, J. Exp. Med.193(9): 1087-1096; Kudlacz et. al., 2008, Eur. J. Pharmacol. 582(1-3):154-161).

Biochemical and genetic studies have shown an association between JAK2and single-chain (e.g., EPO), IL-3 and interferon gamma cytokinereceptor families (Kisseleva et al., 2002, Gene 285:1-24; Levy et al.,2005, Nat. Rev. Mol. Cell Biol. 3:651-662; O'Shea et al., 2002, Cell,109 (suppl.): S121-S131). Consistent with this, JAK2 knockout mice dieof anemia (O'Shea et al., 2002, Cell, 109 (suppl.): S121-S131). Kinaseactivating mutations in JAK2 (e.g., JAK2 V617F) are associated withmyeloproliferative disorders in humans. Additionally, JAK2 associateswith the receptors for cytokines such as IL-5 and Thymic stromallymphopoietin (TSLP). IL-5 is the key cytokine responsible foreosinophil differentiation, growth, activation, survival, andrecruitment to airways (Pelaia et al., 2019, Front. Physiol., 10: 1514;Stirling et al., 2001, Am. J. Respir. Crit. Care Med., 164: 1403-9;Fulkerson and Rothenberg, 2013, Nat. Rev. Drug Discov., 12: 117-9.;Varricchi and Canonica, 2016, Expert. Rev. Clin. Immunol., 12: 903-5).Three monoclonal antibody drugs targeting either IL-5 (Mepolizumab,Reslizumab) or the alpha chain of its receptor (Benralizumab) have beenapproved as treatments for asthma with an eosinophilic phenotype. TSLPis an epithelial-cell-derived cytokine that plays an important role inthe regulation of type II immunity and serves as an alarmin upstream ofTH2 cytokine production (Kitajima et al., 2011, Eur J Immunol., 41:1862-71). Tezepelumab is an antagonist antibody to TSLP. Results from aphase 2 trial indicate it successfully reduced asthma exacerbations inpatients both with and without Type 2-high signatures (Corren et al.,2017, 377: 936-46).

JAK3 associates exclusively with the gamma common cytokine receptorchain, which is present in the IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21cytokine receptor complexes. JAK3 is critical for lymphoid celldevelopment and proliferation and mutations in JAK3 result in severecombined immunodeficiency (SCID) (O'Shea et al., 2002, Cell, 109(suppl.): S121-S131). Based on its role in regulating lymphocytes, JAK3and JAK3-mediated pathways have been targeted for immunosuppressiveindications (e.g., transplantation rejection and rheumatoid arthritis)(Baslund et al., 2005, Arthritis & Rheumatism 52:2686-2692; Changelianet al., 2003, Science 302: 875-878).

TYK2 associates with the type I interferon (e.g., IFNalpha), IL-6,IL-10, IL-12 and IL-23 cytokine receptor complexes (Kisseleva et al.,2002, Gene 285:1-24; Watford, W. T. & O'Shea, J. J., 2006, Immunity25:695-697). Consistent with this, primary cells derived from a TYK2deficient human are defective in type I interferon, IL-6, IL-10, IL-12and IL-23 signaling. A fully human monoclonal antibody targeting theshared p40 subunit of the IL-12 and IL-23 cytokines (Ustekinumab) wasrecently approved by the European Commission for the treatment ofmoderate-to-severe plaque psoriasis (Krueger et al., 2007, N. Engl. J.Med. 356:580-92; Reich et al., 2009, Nat. Rev. Drug Discov. 8:355-356).In addition, an antibody targeting the IL-12 and IL-23 pathwaysunderwent clinical trials for treating Crohn's Disease (Mannon et al.,2004, N. Engl. J. Med. 351:2069-79).

International Patent Application Publication Numbers WO 2010/051549, WO2011/003065, WO 2015/177326 and WO 2017/089390 discuss certainpyrazolopyrimidine compounds that are reported to useful as inhibitorsof one or more Janus kinases. Data for certain specific compoundsshowing inhibition of JAK1 as well as JAK2, JAK3, and/or TYK2 kinases ispresented therein.

Currently there remains a need for additional compounds that areinhibitors of Janus kinases. For example, there is a need for compoundsthat possess useful potency as inhibitors of one or more Janus kinases(e.g., JAK1 and JAK2)-in combination with other pharmacologicalproperties that are necessary to achieve a useful therapeutic benefit.For example, there is a need for potent compounds that demonstrateselectivity for one Janus kinase over other kinases in general (e.g.,selectivity for JAK1 and/or JAK2 over other kinases such as leucine-richrepeat kinase 2 (LRRK2)). There is also a need for potent compounds thatdemonstrate selectivity for one Janus kinase over other Janus kinases(e.g., selectivity for JAK1 and/or JAK2 over JAK3 and/or TYK2).Compounds demonstrating selectivity for both JAK1 and JAK2 over JAK3 andTYK2 could provide a therapeutic benefit, in conditions responsive tothe inhibition of JAK1. Additionally there is currently a need forpotent JAK1 inhibitors that possess other properties (e.g., meltingpoint, pK, solubility, etc.) necessary for formulation andadministration by inhalation. Such compounds would be particularlyuseful for treating conditions such as, for example, asthma.

There accordingly exists a need in the art for additional or alternativetreatments of conditions mediated by JAK kinases, such as thosedescribed above. There is in particular a need for JAK1 and JAK2 kinaseinhibitors usable for inhaled delivery in the treatment of airwayinflammation indications such as asthma.

SUMMARY OF THE INVENTION

Provided herein are pyrazolopyrimidines that inhibit JAK kinases, suchas selected from a compound of Formula (I) a stereoisomer or saltthereof, such as a pharmaceutically acceptable salt thereof. The JAKkinase may be JAK1, JAK2, or both.

One embodiment provides a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   R¹ is: C₁₋₆alkyl; cyano-C₁₋₆alkyl; C₁₋₆alkoxy-(CO)—;        —(CHR^(a))_(m)—NR^(b)R^(c); or —(CHR^(a))_(n)-het¹;    -   R² is: C₁₋₆alkyl; hydroxy-C₁₋₆alkyl; halo-C₁₋₆alkyl;        C₁₋₆alkoxy-C₁₋₆alkyl; C₃₋₆ cycloalkyl;        —(CHR^(a))_(p)—NR^(b)R^(c); het²; —(CHR^(a))_(q)-het³; or phenyl        which may be unsubstituted or substituted once or twice with        R^(d);    -   R³ is: hydrogen; amino; or C₁₋₆ alkyl;    -   R⁴ is: hydrogen; or C₁₋₆alkyl;    -   R⁵ is: hydrogen; or C₁₋₆alkyl;    -   R⁶ is: hydrogen; C₁₋₆alkyl; or R² and R⁶ together with the atoms        to which they are attached may form a six membered ring;    -   m is from 2 to 3;    -   n is from 0 to 2;    -   p is from 0 to 2;    -   each R^(a) is independently: hydrogen; or C₁₋₆ alkyl;    -   each R^(b) is independently: hydrogen; or C₁₋₆ alkyl;    -   each R^(c) is independently: hydrogen; or C₁₋₆alkyl;    -   het¹ is: tetrahydrofuranyl; azetidinyl; or pyrrolidinyl, each of        which may be unsubstituted or substituted once or twice with        R^(e);    -   het² is: pyridinyl; pyrimidinyl; pyrazolyl; imidazolyl; or        isoquinolinyl which may be partially saturated; each of which        may be unsubstituted or substituted once or twice with R^(f);    -   het³ is: azetidinyl; pyrrolidinyl; oxetanyl; or piperidinyl;        each of which may be unsubstituted or substituted once with        R^(g);    -   each R^(d) is independently: C₁₋₆ alkyl; hydroxy; C₁₋₆        alkoxy-C₁₋₆ alkyl; —(CHR^(a))_(q)—NR^(b)R^(c); or phenyl;    -   each R^(e) is independently: C₁₋₆alkyl; or oxo;    -   each R^(f) is independently: C₁₋₆ alkyl; hydroxy-C₁₋₆ alkyl;        oxo; —(CHR^(a))_(r)—NR^(b)R^(c); —(CHR^(a))_(s)-het⁴;    -   each R^(g) is independently: C₁₋₆ alkyl; or acetyl;    -   q is from 1 to 2;    -   r is from 2 to 3;    -   s is from 2 to 3; and    -   het⁴ is: azetidin-1-yl; 1-methyl-azetidin-3-yl; quinuclidinyl;        1-methyl-pyrrolidin-2-yl; or 4-methylpiperazin-1-yl.

Also provided is a pharmaceutical composition comprising a JAK inhibitoras described herein, or a pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable carrier, dilient or excipient.

Also provided is the use of a JAK inhibitor as described herein, or apharmaceutically acceptable salt thereof in therapy, such as in thetreatment of an inflammatory disease (e.g., asthma). Also provided isthe use of a JAK inhibitor as described herein or a pharmaceuticallyacceptable salt thereof for the preparation of a medicament for thetreatment of an inflammatory disease. Also provided is a method ofpreventing, treating or lessening the severity of a disease or conditionresponsive to the inhibition of a Janus kinase activity in a patient,comprising administering to the patient a therapeutically effectiveamount of a JAK inhibitor as described herein or a pharmaceuticallyacceptable salt thereof.

The most validated cytokines in asthma (IL-4, IL-5, IL-9, IL-13, andTSLP) all signal through JAK1 and/or JAK2. The compounds of theinvention are active for both JAK1 and JAK2. Certain of these compoundsoptimally have well-balanced co-activity for both JAK1 and JAK2, or haveslightly higher affinity for JAK1 over JAK2, rather than having a muchgreater activity for one of these kinases over the other. The subjectcompounds also have good selectivity against off-target kinases such asLRRK2, which has been associated with pulmonary toxicity.

While many compounds may exhibit high affinity for both JAK1 and JAK2 insimple biochemical assays, not all such compounds are effective atmediating the relevant cytokines associated with JAK1 and JAK2. Certaincompounds of the invention, in addition to being active for both JAK1and JAK2, are also shown in cell-based assays to be effective atmediation of asthma-relevant cytokines associated with JAK1 and JAK2.

Compounds of the invention also exhibit favorable pharmacokinetic (PK)properties in lung tissue and are useful for inhaled therapies. Whendosed via the inhaled route using techniques such as dry powderinhalation (DPI) or intranasal (IN) delivery, certain compoundsunexpectedly show sustained retention within the lung tissue, with muchlower concentrations in systemic circulation. Such improved PKproperties can advantageously result in smaller dosages and lessfrequent dosing requirements for effective therapies. Certain compoundsexhibit unexpected improved solubility, again providing improvedefficacy in lung. Certain compounds of the invention also exhibitunexpected reduction in cytotoxicity in comparison to other JAKinhibitors.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Halogen” or “halo” refers to F, Cl, Br or I. Additionally, terms suchas “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl,wherein one or more halogens replace a hydrogen(s) of an alkyl group.

The term “alkyl” refers to a saturated linear or branched-chainmonovalent hydrocarbon radical, wherein the alkyl radical may beoptionally substituted. In one example, the alkyl radical is one toeighteen carbon atoms (C₁-C₁₈). In other examples, the alkyl radical isC₀-C₆, C₀-C₅, C₀-C₃, C₁-C₁₀, C₁-C₈, C₁-C₆, C₁-C₅, C₁-C₄, or C₁-C₃. C₀alkyl refers to a bond. Examples of alkyl groups include methyl (Me,—CH₃), ethyl (Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃),2-propyl (i-Pr, i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl,—CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl(s-Bu, s-butyl, —CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl,—C(CH₃)₃), 1-pentyl (n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl(—CH(CH₃)CH₂CH₂CH₃), 3-pentyl (—CH(CH₂CH₃)₂), 2-methyl-2-butyl(—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl (—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl(—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl (—CH₂CH(CH₃)CH₂CH₃), 1-hexyl(—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl (—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl(—CH(CH₂CH₃)(CH₂CH₂CH₃)), 2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃),3-methyl-2-pentyl (—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl(—CH(CH₃)CH₂CH(CH₃)₂), 3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂),2-methyl-3-pentyl (—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl(—C(CH₃)₂CH(CH₃)₂), 3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl and1-octyl. In some embodiments, substituents for “optionally substitutedalkyls” include one to four instances of F, Cl, Br, I, OH, SH, CN, NH₂,NHCH₃, N(CH₃)₂, NO₂, N₃, C(O)CH₃, COOH, CO₂CH₃, methyl, ethyl, propyl,iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo,trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino,SO, SO₂, phenyl, piperidinyl, piperizinyl, and pyrimidinyl, wherein thealkyl, phenyl and heterocyclic portions thereof may be optionallysubstituted, such as by one to four instances of substituents selectedfrom this same list.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical with at least one site of unsaturation, i.e., acarbon-carbon double bond, wherein the alkenyl radical may be optionallysubstituted, and includes radicals having “cis” and “trans”orientations, or alternatively, “E” and “Z” orientations. In oneexample, the alkenyl radical is two to eighteen carbon atoms (C₂-C₁₈).In other examples, the alkenyl radical is C₂-C₁₂, C₂-C₁₀, C₂-C₈, C₂-C₆or C₂-C₃. Examples include, but are not limited to, ethenyl or vinyl(—CH═CH₂), prop-1-enyl (—CH═CHCH₃), prop-2-enyl (—CH₂CH═CH₂),2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl,buta-1,3-dienyl, 2-methylbuta-1,3-diene, hex-1-enyl, hex-2-enyl,hex-3-enyl, hex-4-enyl and hexa-1,3-dienyl. In some embodiments,substituents for “optionally substituted alkenyls” include one to fourinstances of F, Cl, Br, I, OH, SH, CN, NH₂, NHCH₃, N(CH₃)₂, NO₂, N₃,C(O)CH₃, COOH, CO₂CH₃, methyl, ethyl, propyl, iso-propyl, butyl,isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl,difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO₂, phenyl,piperidinyl, piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl andheterocyclic portions thereof may be optionally substituted, such as byone to four instances of substituents selected from this same list.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical with at least one site of unsaturation, i.e., a carbon-carbon,triple bond, wherein the alkynyl radical may be optionally substituted.In one example, the alkynyl radical is two to eighteen carbon atoms(C₂-C₁₈). In other examples, the alkynyl radical is C₂-C₁₂, C₂-C₁₀,C₂-C₈, C₂-C₆ or C₂-C₃. Examples include, but are not limited to, ethynyl(—CCH), prop-1-ynyl (—C≡CCH₃), prop-2-ynyl (propargyl, —CH₂C≡CH),but-1-ynyl, but-2-ynyl and but-3-ynyl. In some embodiments, substituentsfor “optionally substituted alkynyls” include one to four instances ofF, Cl, Br, I, OH, SH, CN, NH₂, NHCH₃, N(CH₃)₂, NO₂, N₃, C(O)CH₃, COOH,CO₂CH₃, methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl,methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl,sulfonylamino, methanesulfonylamino, SO, SO₂, phenyl, piperidinyl,piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl and heterocyclicportions thereof may be optionally substituted, such as by one to fourinstances of substituents selected from this same list.

“Alkylene” refers to a saturated, branched or straight chain hydrocarbongroup having two monovalent radical centers derived by the removal oftwo hydrogen atoms from the same or two different carbon atoms of aparent alkane. In one example, the divalent alkylene group is one toeighteen carbon atoms (C₁-C₁₈). In other examples, the divalent alkylenegroup is C₀-C₆, C₀-C₅, C₀-C₃, C₁-C₁₀, C₁-C₈, C₁-C₆, C₁-C₅, C₁-C₄, orC₁-C₃. The group C₀ alkylene refers to a bond. Example alkylene groupsinclude methylene (—CH₂—), 1,1-ethyl (—CH(CH₃)—), (1,2-ethyl (—CH₂CH₂—),1,1-propyl (—CH(CH₂CH₃)—), 2,2-propyl (—C(CH₃)₂—), 1,2-propyl(—CH(CH₃)CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,1-dimethyleth-1,2-yl(—C(CH₃)₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), and the like.

The term “heteroalkyl” refers to a straight or branched chain monovalenthydrocarbon radical, consisting of the stated number of carbon atoms,or, if none are stated, up to 18 carbon atoms, and from one to fiveheteroatoms selected from the group consisting of O, N, Si and S, andwherein the nitrogen and sulfur atoms can optionally be oxidized and thenitrogen heteroatom can optionally be quaternized. In some embodiments,the heteroatom is selected from O, N and S, wherein the nitrogen andsulfur atoms can optionally be oxidized and the nitrogen heteroatom canoptionally be quaternized. The heteroatom(s) can be placed at anyinterior position of the heteroalkyl group, including the position atwhich the alkyl group is attached to the remainder of the molecule(e.g., —O—CH₂—CH₃). Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃,—Si(CH₃)₃ and —CH₂—CH═N—OCH₃. Up to two heteroatoms can be consecutive,such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Heteroalkylgroups can be optionally substituted. In some embodiments, substituentsfor “optionally substituted heteroalkyls” include one to four instancesof F, Cl, Br, I, OH, SH, CN, NH₂, NHCH₃, N(CH₃)₂, NO₂, N₃, C(O)CH₃,COOH, CO₂CH₃, methyl, ethyl, propyl, iso-propyl, butyl, isobutyl,cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl,difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO₂, phenyl,piperidinyl, piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl andheterocyclic portions thereof may be optionally substituted, such as byone to four instances of substituents selected from this same list.

“Amino” means primary (i.e., —NH₂), secondary (i.e., —NRH), tertiary(i.e., —NRR) and quaternary (i.e., —N(+)RRR) amines, that are optionallysubstituted, in which each R is the same or different and selected fromalkyl, cycloalkyl, aryl, and heterocyclyl, wherein the alkyl,cycloalkyl, aryl and heterocyclyl groups are as defined herein.Particular secondary and tertiary amines are alkylamine, dialkylamine,arylamine, diarylamine, aralkylamine and diaralkylamine, wherein thealkyl and aryl portions can be optionally substituted. Particularsecondary and tertiary amines are methylamine, ethylamine, propylamine,isopropylamine, phenylamine, benzylamine, dimethylamine, diethylamine,dipropylamine and diisopropylamine. In some embodiments, R groups of aquarternary amine are each independently optionally substituted alkylgroups.

“Aryl” refers to a carbocyclic aromatic group, whether or not fused toone or more groups, having the number of carbon atoms designated, or ifno number is designated, up to 14 carbon atoms. One example includesaryl groups having 6-14 carbon atoms. Another example includes arylgroups having 6-10 carbon atoms. Examples of aryl groups include phenyl,naphthyl, biphenyl, phenanthrenyl, naphthacenyl,1,2,3,4-tetrahydronaphthalenyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, andthe like (see, e.g., Lang's Handbook of Chemistry (Dean, J. A., ed.)13th ed. Table 7-2 [1985]). A particular aryl is phenyl. Substitutedphenyl or substituted aryl means a phenyl group or aryl groupsubstituted with one, two, three, four or five substituents, forexample, 1-2, 1-3 or 1-4 substituents, such as chosen from groupsspecified herein (see “optionally substituted” definition), such as F,Cl, Br, I, OH, SH, CN, NH₂, NHCH₃, N(CH₃)₂, NO₂, N₃, C(O)CH₃, COOH,CO₂CH₃, methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl,methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl,sulfonylamino, methanesulfonylamino, SO, SO₂, phenyl, piperidinyl,piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl and heterocyclicportions thereof may be optionally substituted, such as by one to fourinstances of substituents selected from this same list. Examples of theterm “substituted phenyl” include a mono- or di(halo)phenyl group suchas 2-chlorophenyl, 2-bromophenyl, 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl,4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl,2-fluorophenyl, 2,4-difluorophenyl and the like; a mono- ordi(hydroxy)phenyl group such as 4-hydroxyphenyl, 3-hydroxyphenyl,2,4-dihydroxyphenyl, the protected-hydroxy derivatives thereof and thelike; a nitrophenyl group such as 3- or 4-nitrophenyl; a cyanophenylgroup, for example, 4-cyanophenyl; a mono- or di(alkyl)phenyl group suchas 4-methylphenyl, 2,4-dimethylphenyl, 2-methylphenyl,4-(isopropyl)phenyl, 4-ethylphenyl, 3-(n-propyl)phenyl and the like; amono or di(alkoxy)phenyl group, for example, 3,4-dimethoxyphenyl,3-methoxy-4-benzyloxyphenyl, 3-ethoxyphenyl, 4-(isopropoxy)phenyl,4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl and the like; 3- or4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protectedcarboxy)phenyl group such 4-carboxyphenyl, a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as3-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono-or di(N-(methylsulfonylamino))phenyl such as3-(N-methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups where the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl, 2-chloro-5-difluoromethoxy and the like, aswell as trisubstituted phenyl groups where the substituents aredifferent, for example 3-methoxy-4-benzyloxy-6-methyl sulfonylamino,3-methoxy-4-benzyloxy-6-phenyl sulfonylamino, and tetrasubstitutedphenyl groups where the substituents are different such as3-methoxy-4-benzyloxy-5-methyl-6-phenyl sulfonylamino. In someembodiments, a substituent of an aryl, such as phenyl, comprises anamide. For example, an aryl (e.g., phenyl) substituent may be —(CH₂)₀₋₄CONR′R″, wherein R′ and R″ each independently refer to groups including,for example, hydrogen; unsubstituted C₁-C₆alkyl; C₁-C₆alkyl substitutedby halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆alkoxy, oxo or NR′R″; unsubstituted C₁-C₆ heteroalkyl; C₁-C₆ heteroalkylsubstituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstitutedC₁-C₆ alkoxy, oxo or NR′R″; unsubstituted C₆-C₁₀ aryl; C₆-C₁₀ arylsubstituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstitutedC₁-C₆ alkoxy, or NR′R″; unsubstituted 3-11 membered heterocyclyl (e.g.,5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, Nand S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatomsselected from O, N and S); and 3-11 membered heterocyclyl (e.g., 5-6membered heteroaryl containing 1 to 4 heteroatoms selected from O, N andS or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatomsselected from O, N and S) substituted by halogen, OH, CN, unsubstitutedC₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″; or R′ and R″ canbe combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or7-membered ring wherein a ring atom is optionally substituted with N, Oor S and wherein the ring is optionally substituted with halogen, OH,CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″.

“Cycloalkyl” refers to a non-aromatic, saturated or partiallyunsaturated hydrocarbon ring group wherein the cycloalkyl group may beoptionally substituted independently with one or more substituentsdescribed herein. In one example, the cycloalkyl group is 3 to 12 carbonatoms (C₃-C₁₂). In other examples, cycloalkyl is C₃-C₈, C₃-C₁₀ orC₅-C₁₀. In other examples, the cycloalkyl group, as a monocycle, isC₃-C₈, C₃-C₆ or C₅-C₆. In another example, the cycloalkyl group, as abicycle, is C₇-C₁₂. In another example, the cycloalkyl group, as a spirosystem, is C₅-C₁₂. Examples of monocyclic cycloalkyl includecyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,perdeuteriocyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl,1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl,cyclodecyl, cycloundecyl and cyclododecyl. Exemplary arrangements ofbicyclic cycloalkyls having 7 to 12 ring atoms include, but are notlimited to, [4,4], [4,5], [5,5], [5,6] or [6,6] ring systems. Exemplarybridged bicyclic cycloalkyls include, but are not limited to,bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.Examples of spiro cycloalkyl include, spiro[2.2]pentane,spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane andspiro[4.5]decane. In some embodiments, substituents for “optionallysubstituted cycloalkyls” include one to four instances of F, Cl, Br, I,OH, SH, CN, NH₂, NHCH₃, N(CH₃)₂, NO₂, N₃, C(O)CH₃, COOH, CO₂CH₃, methyl,ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy,ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino,methanesulfonylamino, SO, SO₂, phenyl, piperidinyl, piperizinyl, andpyrimidinyl, wherein the alkyl, aryl and heterocyclic portions thereofmay be optionally substituted, such as by one to four instances ofsubstituents selected from this same list. In some embodiments, asubstituent of a cycloalkyl comprises an amide. For example, acycloalkyl substituent may be —(CH₂)₀₋₄ CONR′R″, wherein R′ and R″ eachindependently refer to groups including, for example, hydrogen;unsubstituted C₁-C₆alkyl; C₁-C₆alkyl substituted by halogen, OH, CN,unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″;unsubstituted C₁-C₆ heteroalkyl; C₁-C₆ heteroalkyl substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,oxo or NR′R″; unsubstituted C₆-C₁₀ aryl; C₆-C₁₀ aryl substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,or NR′R″; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 memberedheteroaryl containing 1 to 4 heteroatoms selected from O, N and S or4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selectedfrom O, N and S); and 3-11 membered heterocyclyl (e.g., 5-6 memberedheteroaryl containing 1 to 4 heteroatoms selected from O, N and S or4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selectedfrom O, N and S) substituted by halogen, OH, CN, unsubstitutedC₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″; or R′ and R″ canbe combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or7-membered ring wherein a ring atom is optionally substituted with N, Oor S and wherein the ring is optionally substituted with halogen, OH,CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″.

“Heterocyclic group”, “heterocyclic”, “heterocycle”, “heterocyclyl”, or“heterocyclo” are used interchangeably and refer to any mono-, bi-,tricyclic or spiro, saturated or unsaturated, aromatic (heteroaryl) ornon-aromatic (e.g., heterocycloalkyl), ring system, having 3 to 20 ringatoms (e.g., 3-10 ring atoms), where the ring atoms are carbon, and atleast one atom in the ring or ring system is a heteroatom selected fromnitrogen, sulfur or oxygen. If any ring atom of a cyclic system is aheteroatom, that system is a heterocycle, regardless of the point ofattachment of the cyclic system to the rest of the molecule. In oneexample, heterocyclyl includes 3-11 ring atoms (“members”) and includesmonocycles, bicycles, tricycles and spiro ring systems, wherein the ringatoms are carbon, where at least one atom in the ring or ring system isa heteroatom selected from nitrogen, sulfur or oxygen. In one example,heterocyclyl includes 1 to 4 heteroatoms. In one example, heterocyclylincludes 1 to 3 heteroatoms. In another example, heterocyclyl includes3- to 7-membered monocycles having 1-2, 1-3 or 1-4 heteroatoms selectedfrom nitrogen, sulfur or oxygen. In another example, heterocyclylincludes 4- to 6-membered monocycles having 1-2, 1-3 or 1-4 heteroatomsselected from nitrogen, sulfur or oxygen. In another example,heterocyclyl includes 3-membered monocycles. In another example,heterocyclyl includes 4-membered monocycles. In another example,heterocyclyl includes 5-6 membered monocycles, e.g., 5-6 memberedheteroaryl. In another example, heterocyclyl includes 3-11 memberedheterocycloyalkyls, such as 4-11 membered heterocycloalkyls. In someembodiments, a heterocycloalkyl includes at least one nitrogen. In oneexample, the heterocyclyl group includes 0 to 3 double bonds. Anynitrogen or sulfur heteroatom may optionally be oxidized (e.g., NO, SO,SO₂), and any nitrogen heteroatom may optionally be quaternized (e.g.,[NR₄]⁺Cl⁻, [NR₄]⁺OH⁻). Example heterocycles are oxiranyl, aziridinyl,thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl,1,3-dithietanyl, pyrrolidinyl, dihydro-1H-pyrrolyl, dihydrofuranyl,tetrahydrofuranyl, dihydrothienyl, tetrahydrothienyl, imidazolidinyl,piperidinyl, piperazinyl, isoquinolinyl, tetrahydroisoquinolinyl,morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl,tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl,oxazinanyl, thiazinanyl, thioxanyl, homopiperazinyl, homopiperidinyl,azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl,1,4-diazepanyl, diazepinyl, thiazepinyl, thiazepanyl,tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl,1,1-dioxoisothiazolidinonyl, oxazolidinonyl, imidazolidinonyl,4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzoimidazolyl,4,5,6,7-tetrahydrobenzo[d]imidazolyl,1,6-dihydroimidazol[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl, oxazinyl,thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl,thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl,dihydropyrimidyl, tetrahydropyrimidyl, 1-pyrrolinyl, 2-pyrrolinyl,3-pyrrolinyl, indolinyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl,1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl,pyrimidinonyl, pyrimidindionyl, pyrimidin-2,4-dionyl, piperazinonyl,piperazindionyl, pyrazolidinylimidazolinyl, 3-azabicyclo[3.1.0]hexanyl,3,6-diazabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl,3-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[4.1.0]heptanyl,azabicyclo[2.2.2]hexanyl, 2-azabicyclo[3.2.1]octanyl,8-azabicyclo[3.2.1]octanyl, 2-azabicyclo[2.2.2]octanyl,8-azabicyclo[2.2.2]octanyl, 7-oxabicyclo[2.2.1]heptane,azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decanyl,1-azaspiro[4.5]decan-2-only, azaspiro[5.5]undecanyl, tetrahydroindolyl,octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl,1,1-dioxohexahydrothiopyranyl. Examples of 5-membered heterocyclescontaining a sulfur or oxygen atom and one to three nitrogen atoms arethiazolyl, including thiazol-2-yl and thiazol-2-yl N-oxide,thiadiazolyl, including 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl,oxazolyl, for example oxazol-2-yl, and oxadiazolyl, such as1,3,4-oxadiazol-5-yl, and 1,2,4-oxadiazol-5-yl. Example 5-membered ringheterocycles containing 2 to 4 nitrogen atoms include imidazolyl, suchas imidazol-2-yl; triazolyl, such as 1,3,4-triazol-5-yl;1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyl, such as1H-tetrazol-5-yl. Example benzo-fused 5-membered heterocycles arebenzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl. Example6-membered heterocycles contain one to three nitrogen atoms andoptionally a sulfur or oxygen atom, for example pyridyl, such aspyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyl, such as pyrimid-2-yland pyrimid-4-yl; triazinyl, such as 1,3,4-triazin-2-yl and1,3,5-triazin-4-yl; pyridazinyl, in particular pyridazin-3-yl, andpyrazinyl. The pyridine N-oxides and pyridazine N-oxides and thepyridyl, pyrimid-2-yl, pyrimid-4-yl, pyridazinyl and the1,3,4-triazin-2-yl groups, are other example heterocycle groups.Heterocycles may be optionally substituted. For example, substituentsfor “optionally substituted heterocycles” include one to four instancesof F, Cl, Br, I, OH, SH, CN, NH₂, NHCH₃, N(CH₃)₂, NO₂, N₃, C(O)CH₃,COOH, CO₂CH₃, methyl, ethyl, propyl, iso-propyl, butyl, isobutyl,cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl,difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO₂, phenyl,piperidinyl, piperizinyl, and pyrimidinyl, wherein the alkyl, aryl andheterocyclic portions thereof may be optionally substituted, such as byone to four instances of substituents selected from this same list. Insome embodiments, a substituent of a heterocyclic group, such as aheteroaryl or heterocycloalkyl, comprises an amide. For example, aheterocyclic (e.g., heteroaryl or heterocycloalkyl) substituent may be—(CH₂)₀₋₄CONR′R″, wherein R′ and R″ each independently refer to groupsincluding, for example, hydrogen; unsubstituted C₁-C₆alkyl; C₁-C₆alkylsubstituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstitutedC₁-C₆ alkoxy, oxo or NR′R″; unsubstituted C₁-C₆ heteroalkyl; C₁-C₆heteroalkyl substituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl,unsubstituted C₁-C₆ alkoxy, oxo or NR′R″; unsubstituted C₆-C₁₀ aryl;C₆-C₁₀ aryl substituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl,unsubstituted C₁-C₆ alkoxy, or NR′R″; unsubstituted 3-11 memberedheterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4heteroatoms selected from O, N and S or 4-11 membered heterocycloalkylcontaining 1 to 4 heteroatoms selected from O, N and S); and 3-11membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4heteroatoms selected from O, N and S or 4-11 membered heterocycloalkylcontaining 1 to 4 heteroatoms selected from O, N and S) substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,oxo or NR′R″; or R′ and R″ can be combined with the nitrogen atom toform a 3-, 4-, 5-, 6-, or 7-membered ring wherein a ring atom isoptionally substituted with N, O or S and wherein the ring is optionallysubstituted with halogen, OH, CN, unsubstituted C₁-C₆alkyl,unsubstituted C₁-C₆ alkoxy, oxo or NR′R″.

“Heteroaryl” refers to any mono-, bi-, or tricyclic ring system where atleast one ring is a 5- or 6-membered aromatic ring containing from 1 to4 heteroatoms selected from nitrogen, oxygen, and sulfur, and in anexample embodiment, at least one heteroatom is nitrogen. See, forexample, Lang's Handbook of Chemistry (Dean, J. A., ed.) 13^(th) ed.Table 7-2 [1985]. Included in the definition are any bicyclic groupswhere any of the above heteroaryl rings are fused to an aryl ring,wherein the aryl ring or the heteroaryl ring is joined to the remainderof the molecule. In one embodiment, heteroaryl includes 5-6 memberedmonocyclic aromatic groups where one or more ring atoms is nitrogen,sulfur or oxygen. Example heteroaryl groups include thienyl, furyl,imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl,tetrazinyl, tetrazolo[1,5-b]pyridazinyl, imidazol[1,2-a]pyrimidinyl andpurinyl, as well as benzo-fused derivatives, for example benzoxazolyl,benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl,benzoimidazolyl and indolyl. Heteroaryl groups can be optionallysubstituted. In some embodiments, substituents for “optionallysubstituted heteroaryls” include one to four instances of F, Cl, Br, I,OH, SH, CN, NH₂, NHCH₃, N(CH₃)₂, NO₂, N₃, C(O)CH₃, COOH, CO₂CH₃, methyl,ethyl, propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy,ethoxy, propoxy, trifluoromethyl, difluoromethyl, sulfonylamino,methanesulfonylamino, SO, SO₂, phenyl, piperidinyl, piperizinyl, andpyrimidinyl, wherein the alkyl, phenyl and heterocyclic portions thereofmay be optionally substituted, such as by one to four instances ofsubstituents selected from this same list. In some embodiments, asubstituent of a heteroaryl comprises an amide. For example, aheteroaryl substituent may be —(CH₂)₀₋₄CONR′R″, wherein R′ and R″ eachindependently refer to groups including, for example, hydrogen;unsubstituted C₁-C₆alkyl; C₁-C₆alkyl substituted by halogen, OH, CN,unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″;unsubstituted C₁-C₆ heteroalkyl; C₁-C₆ heteroalkyl substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,oxo or NR′R″; unsubstituted C₆-C₁₀ aryl; C₆-C₁₀ aryl substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,or NR′R″; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 memberedheteroaryl containing 1 to 4 heteroatoms selected from O, N and S or4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selectedfrom O, N and S); and 3-11 membered heterocyclyl (e.g., 5-6 memberedheteroaryl containing 1 to 4 heteroatoms selected from O, N and S or4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selectedfrom O, N and S) substituted by halogen, OH, CN, unsubstitutedC₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″; or R′ and R″ canbe combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or7-membered ring wherein a ring atom is optionally substituted with N, Oor S and wherein the ring is optionally substituted with halogen, OH,CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″.

In particular embodiments, a heterocyclyl group is attached at a carbonatom of the heterocyclyl group. By way of example, carbon bondedheterocyclyl groups include bonding arrangements at position 2, 3, 4, 5,or 6 of a pyridine ring, position 3, 4, 5, or 6 of a pyridazine ring,position 2, 4, 5, or 6 of a pyrimidine ring, position 2, 3, 5, or 6 of apyrazine ring, position 2, 3, 4, or 5 of a furan, tetrahydrofuran,thiofuran, thiophene, pyrrole or tetrahydropyrrole ring, position 2, 4,or 5 of an oxazole, imidazole or thiazole ring, position 3, 4, or 5 ofan isoxazole, pyrazole, or isothiazole ring, position 2 or 3 of anaziridine ring, position 2, 3, or 4 of an azetidine ring, position 2, 3,4, 5, 6, 7, or 8 of a quinoline ring or position 1, 3, 4, 5, 6, 7, or 8of an isoquinoline ring.

In certain embodiments, the heterocyclyl group is N-attached. By way ofexample, nitrogen bonded heterocyclyl or heteroaryl groups includebonding arrangements at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole,position 2 of a isoindole, or isoindoline, position 4 of a morpholine,and position 9 of a carbazole, or β-carboline.

The term “alkoxy” refers to a linear or branched monovalent radicalrepresented by the formula —OR in which R is alkyl, as defined herein.Alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, mono-, di-and tri-fluoromethoxy and cyclopropoxy.

“Acyl” means a carbonyl containing substituent represented by theformula —C(O)—R in which R is hydrogen, alkyl, cycloalkyl, aryl orheterocyclyl, wherein the alkyl, cycloalkyl, aryl and heterocyclyl areas defined herein. Acyl groups include alkanoyl (e.g., acetyl), aroyl(e.g., benzoyl), and heteroaroyl (e.g., pyridinoyl).

“Optionally substituted” unless otherwise specified means that a groupmay be unsubstituted or substituted by one or more (e.g., 0, 1, 2, 3, 4,or 5 or more, or any range derivable therein) of the substituents listedfor that group in which said substituents may be the same or different.In an embodiment, an optionally substituted group has 1 substituent. Inanother embodiment an optionally substituted group has 2 substituents.In another embodiment an optionally substituted group has 3substituents. In another embodiment an optionally substituted group has4 substituents. In another embodiment an optionally substituted grouphas 5 substituents.

Optional substituents for alkyl radicals, alone or as part of anothersubstituent (e.g., alkoxy), as well as alkylenyl, alkenyl, alkynyl,heteroalkyl, heterocycloalkyl, and cycloalkyl, also each alone or aspart of another substituent, can be a variety of groups, such as thosedescribed herein, as well as selected from the group consisting ofhalogen; oxo; CN; NO; N₃; —OR; perfluoro-C₁-C₄ alkoxy; unsubstitutedC₃-C₇ cycloalkyl; C₃-C₇ cycloalkyl substituted by halogen, OH, CN,unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″;unsubstituted C₆-C₁₀ aryl (e.g., phenyl); C₆-C₁₀ aryl substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,or NR′R″; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6 memberedheteroaryl containing 1 to 4 heteroatoms selected from O, N and S or4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selectedfrom O, N and S); 3-11 membered heterocyclyl (e.g., 5-6 memberedheteroaryl containing 1 to 4 heteroatoms selected from O, N and S or4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms selectedfrom O, N and S) substituted by halogen, OH, CN, unsubstitutedC₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or

NR′R″; —NR′R″; —SR′; —SiR′R″R″; —OC(O)R′; —C(O)R′; —CO₂R; —CONR′R″;—OC(O)NR′R″; —NR″C(O)R; —NR′″ C(O)NR′R″; —NR″C(O)₂R; —S(O)₂R;—S(O)₂NR′R″; —NR'S(O)₂R″; —NR″'S(O)₂NR′R″; amidinyl; guanidinyl;—(CH₂)₁₋₄—OR; —(CH₂)₁₄—NR′R″; —(CH₂)₁₋₄—SR; —(CH₂)₁₋₄—SiR′R″R″;—(CH₂)₁₋₄—OC(O)R; —(CH₂)₁₋₄—C(O)R; —(CH₂)₁₋₄—CO₂R; and —(CH₂)₁₋₄CONR′R″, or combinations thereof, in a number ranging from zero to(2m′+1), where m′ is the total number of carbon atoms in such radical.R′, R″ and R′″ each independently refer to groups including, forexample, hydrogen; unsubstituted C₁-C₆alkyl; C₁-C₆alkyl substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,oxo or NR′R″; unsubstituted C₁-C₆ heteroalkyl; C₁-C₆ heteroalkylsubstituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstitutedC₁-C₆ alkoxy, oxo or NR′R″; unsubstituted C₆-C₁₀ aryl; C₆-C₁₀ arylsubstituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstitutedC₁-C₆ alkoxy, or NR′R″; unsubstituted 3-11 membered heterocyclyl (e.g.,5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, Nand S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatomsselected from O, N and S); and 3-11 membered heterocyclyl (e.g., 5-6membered heteroaryl containing 1 to 4 heteroatoms selected from O, N andS or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatomsselected from O, N and S) substituted by halogen, OH, CN, unsubstitutedC₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring wherein aring atom is optionally substituted with N, O or S and wherein the ringis optionally substituted with halogen, OH, CN, unsubstitutedC₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″. For example,—NR′R″ is meant to include 1-pyrrolidinyl and 4-morpholinyl.

Similarly, optional substituents for the aryl and heteroaryl groups arevaried. In some embodiments, substituents for aryl and heteroaryl groupsare selected from the group consisting of halogen; CN; NO; N₃; —OR;perfluoro-C₁_C₄ alkoxy; unsubstituted C₃-C₇ cycloalkyl; C₃-C₇ cycloalkylsubstituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstitutedC₁-C₆ alkoxy, oxo or NR′R″; unsubstituted C₆-C₁₀ aryl (e.g., phenyl);C₆-C₁₀ aryl substituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl,unsubstituted C₁-C₆ alkoxy, or NR′R″; unsubstituted 3-11 memberedheterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4heteroatoms selected from O, N and S or 4-11 membered heterocycloalkylcontaining 1 to 4 heteroatoms selected from O, N and S); 3-11 memberedheterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4heteroatoms selected from O, N and S or 4-11 membered heterocycloalkylcontaining 1 to 4 heteroatoms selected from O, N and S) substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,oxo or

NR′R″; —NR′R″; —SR′; —SiR′R″R″; —OC(O)R′; —C(O)R′; —CO₂R; —CONR′R″;—OC(O)NR′R″; —NR″C(O)R; —NR′″ C(O)NR′R″; —NR″C(O)₂R; —S(O)₂R;—S(O)₂NR′R″; —NR'S(O)₂R″; —NR″'S(O)₂NR′R″; amidinyl; guanidinyl;—(CH₂)₁₋₄—OR; —(CH₂)₁₋₄—NR′R″; —(CH₂)₁₋₄—SR; —(CH₂)₁₋₄—SiR′R″R″;—(CH₂)₁₋₄—OC(O)R; —(CH₂)₁₋₄—C(O)R; —(CH₂)₁₋₄—CO₂R; and —(CH₂)₁₋₄CONR′R″, or combinations thereof, in a number ranging from zero to(2m′+1), where m′ is the total number of carbon atoms in such radical.R′, R″ and R′″ each independently refer to groups including, forexample, hydrogen; unsubstituted C₁-C₆alkyl; C₁-C₆alkyl substituted byhalogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy,oxo or NR′R″; unsubstituted C₁-C₆ heteroalkyl; C₁-C₆ heteroalkylsubstituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstitutedC₁-C₆ alkoxy, oxo or NR′R″; unsubstituted C₆-C₁₀ aryl; C₆-C₁₀ arylsubstituted by halogen, OH, CN, unsubstituted C₁-C₆alkyl, unsubstitutedC₁-C₆ alkoxy, or NR′R″; unsubstituted 3-11 membered heterocyclyl (e.g.,5-6 membered heteroaryl containing 1 to 4 heteroatoms selected from O, Nand S or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatomsselected from O, N and S); and 3-11 membered heterocyclyl (e.g., 5-6membered heteroaryl containing 1 to 4 heteroatoms selected from O, N andS or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatomsselected from O, N and S) substituted by halogen, OH, CN, unsubstitutedC₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring wherein aring atom is optionally substituted with N, O or S and wherein the ringis optionally substituted with halogen, OH, CN, unsubstitutedC₁-C₆alkyl, unsubstituted C₁-C₆ alkoxy, oxo or NR′R″. For example,—NR′R″ is meant to include 1-pyrrolidinyl and 4-morpholinyl.

The term “oxo” refers to ═O or (═O)₂.

As used herein a wavy line that intersects a bond in a chemicalstructure indicate the point of attachment of the atom to which the wavybond is connected in the chemical structure to the remainder of amolecule, or to the remainder of a fragment of a molecule. In someembodiments, an arrow together with an asterisk is used in the manner ofa wavy line to indicate a point of attachment.

In certain embodiments, divalent groups are described genericallywithout specific bonding configurations. It is understood that thegeneric description is meant to include both bonding configurations,unless specified otherwise. For example, in the group R′—R²—R³, if thegroup R² is described as —CH₂C(O)—, then it is understood that thisgroup can be bonded both as R′—CH₂C(O)—R³, and as R′—C(O)CH₂—R³, unlessspecified otherwise.

The terms “compound(s) of the invention,” and “compound(s) of thepresent invention” and the like, unless otherwise indicated, includecompounds of Formula (I) herein, such as compounds 1-18, sometimesreferred to as JAK inhibitors, including stereoisomers (includingatropisomers), geometric isomers, tautomers, solvates, metabolites,isotopes, salts (e.g., pharmaceutically acceptable salts), and prodrugsthereof. In some embodiments, solvates, metabolites, isotopes orprodrugs are excluded, or any combination thereof.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an adverse, allergic or otheruntoward reaction when administered to an animal, such as, for example,a human, as appropriate.

Compounds of the present invention may be in the form of a salt, such asa pharmaceutically acceptable salt. “Pharmaceutically acceptable salts”include both acid and base addition salts. “Pharmaceutically acceptableacid addition salt” refers to those salts which retain the biologicaleffectiveness and properties of the free bases and which are notbiologically or otherwise undesirable, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid and the like, and organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic, and sulfonic classes of organic acids such asformic acid, acetic acid, propionic acid, glycolic acid, gluconic acid,lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid,maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid,aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoicacid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, salicyclic acid and the like.

“Pharmaceutically acceptable base addition salts” include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. Particular base addition salts are the ammonium, potassium,sodium, calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases include salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particular organicnon-toxic bases include isopropylamine, diethylamine, ethanolamine,tromethamine, dicyclohexylamine, choline, and caffeine.

In some embodiments, a salt is selected from a hydrochloride,hydrobromide, trifluoroacetate, sulphate, phosphate, acetate, fumarate,maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate,methanesulphonate, p-toluenesulphonate, bisulphate, benzenesulphonate,ethanesulphonate, malonate, xinafoate, ascorbate, oleate, nicotinate,saccharinate, adipate, formate, glycolate, palmitate, L-lactate,D-lactate, aspartate, malate, L-tartrate, D-tartrate, stearate, furoate(e.g., 2-furoate or 3-furoate), napadisylate(naphthalene-1,5-disulfonate or naphthalene-1-(sulfonicacid)-5-sulfonate), edisylate (ethane-1,2-disulfonate orethane-1-(sulfonic acid)-2-sulfonate), isethionate(2-hydroxyethylsulfonate), 2-mesitylenesulphonate,2-naphthalenesulphonate, 2,5-dichlorobenzenesulphonate, D-mandelate,L-mandelate, cinnamate, benzoate, adipate, esylate, malonate, mesitylate(2-mesitylenesulphonate), napsylate (2-naphthalenesulfonate), camsylate(camphor-10-sulphonate, for example (1S)-(+)-10-camphorsulfonic acidsalt), glutamate, glutarate, hippurate (2-(benzoylamino)acetate),orotate, xylate (p-xylene-2-sulphonate), and pamoic(2,2′-dihydroxy-1,1′-dinaphthylmethane-3,3′-dicarboxylate).

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

“Stereoisomers” refer to compounds that have identical chemicalconstitution, but differ with regard to the arrangement of the atoms orgroups in space. Stereoisomers include diastereomers, enantiomers,conformers and the like.

“Chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g., melting points,boiling points, spectral properties or biological activities. Mixturesof diastereomers may separate under high resolution analyticalprocedures such as electrophoresis and chromatography such as HPLC.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. Many organic compounds exist in optically active forms,i.e., they have the ability to rotate the plane of plane-polarizedlight. In describing an optically active compound, the prefixes D and L,or R and S, are used to denote the absolute configuration of themolecule about its chiral center(s). The prefixes d and 1 or (+) and (−)are employed to designate the sign of rotation of plane-polarized lightby the compound, with (−) or 1 meaning that the compound islevorotatory. A compound prefixed with (+) or d is dextrorotatory. For agiven chemical structure, these stereoisomers are identical except thatthey are mirror images of one another. A specific stereoisomer may alsobe referred to as an enantiomer, and a mixture of such isomers is oftencalled an enantiomeric mixture. A 50:50 mixture of enantiomers isreferred to as a racemic mixture or a racemate, which may occur wherethere has been no stereoselection or stereospecificity in a chemicalreaction or process. The terms “racemic mixture” and “racemate” refer toan equimolar mixture of two enantiomeric species, devoid of opticalactivity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. A “solvate” refersto an association or complex of one or more solvent molecules and acompound of the present invention. Examples of solvents that formsolvates include water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine. Certain compounds of the presentinvention can exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are intended to be within the scope of thepresent invention. The term “hydrate” refers to the complex where thesolvent molecule is water.

A “metabolite” refers to a product produced through metabolism in thebody of a specified compound or salt thereof. Such products can result,for example, from the oxidation, reduction, hydrolysis, amidation,deamidation, esterification, deesterification, enzymatic cleavage, andthe like, of the administered compound.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it in a detectable dose (e.g., greater than about 0.5mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to ahuman, allowing sufficient time for metabolism to occur (typically about30 seconds to 30 hours) and isolating its conversion products from theurine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

A “subject,” “individual,” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, farm animals (such as cows), sport animals, pets (such asguinea pigs, cats, dogs, rabbits and horses), primates, mice and rats.In certain embodiments, a mammal is a human. In embodiments comprisingadministration of a JAK inhibitor as described herein or apharmaceutically acceptable salt thereof to a patient, the patient maybe in need thereof.

The term “Janus kinase” refers to JAK1, JAK2, JAK3 and TYK2 proteinkinases. In some embodiments, a Janus kinase may be further defined asone of JAK1, JAK2, JAK3 or TYK2. In any embodiment, any one of JAK1,JAK2, JAK3 and TYK2 may be specifically excluded as a Janus kinase. Insome embodiments, a Janus kinase is JAK1. In some embodiments, a Januskinase is a combination of JAK1 and JAK2.

The terms “inhibiting” and “reducing,” or any variation of these terms,includes any measurable decrease or complete inhibition to achieve adesired result. For example, there may be a decrease of about, at mostabout, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or anyrange derivable therein, reduction of activity (e.g., JAK1 activity)compared to normal.

“Therapeutically effective amount” means an amount of a compound or asalt thereof (e.g., a pharmaceutically acceptable salt thereof) of thepresent invention that (i) treats or prevents the particular disease,condition or disorder, or (ii) attenuates, ameliorates or eliminates oneor more symptoms of the particular disease, condition, or disorder, andoptionally (iii) prevents or delays the onset of one or more symptoms ofthe particular disease, condition or disorder described herein. In someembodiments, the therapeutically effective amount is an amountsufficient to decrease or alleviate the symptoms of an autoimmune orinflammatory disease (e.g., asthma). In some embodiments, atherapeutically effective amount is an amount of a chemical entitydescribed herein sufficient to significantly decrease the activity ornumber of B-cells. In the case of cancer, the therapeutically effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; or relieve to some extent one or more of thesymptoms associated with the cancer. To the extent the drug may preventgrowth or kill existing cancer cells, it may be cytostatic or cytotoxic.For cancer therapy, efficacy can, for example, be measured by assessingthe time to disease progression (TTP) or determining the response rate(RR).

“Treatment” (and variations such as “treat” or “treating”) refers toclinical intervention in an attempt to alter the natural course of theindividual or cell being treated, and can be performed either forprophylaxis or during the course of clinical pathology. Desirableeffects of treatment include preventing occurrence or recurrence ofdisease, alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, stabilized (i.e., notworsening) state of disease, decreasing the rate of disease progression,amelioration or palliation of the disease state, prolonging survival ascompared to expected survival if not receiving treatment and remissionor improved prognosis. In some embodiments, a compound of the inventionor a salt thereof (e.g., a pharmaceutically acceptable salt thereof), isused to delay development of a disease or disorder or to slow theprogression of a disease or disorder. Those in need of treatment includethose already with the condition or disorder as well as those prone tohave the condition or disorder, (for example, through a geneticmutation) or those in which the condition or disorder is to beprevented.

“Inflammatory disorder” refers to any disease, disorder or syndrome inwhich an excessive or unregulated inflammatory response leads toexcessive inflammatory symptoms, host tissue damage, or loss of tissuefunction. “Inflammatory disorder” also refers to a pathological statemediated by influx of leukocytes or neutrophil chemotaxis.

“Inflammation” refers to a localized, protective response elicited byinjury or destruction of tissues, which serves to destroy, dilute, orwall off (sequester) both the injurious agent and the injured tissue.Inflammation is notably associated with influx of leukocytes orneutrophil chemotaxis. Inflammation can result from infection withpathogenic organisms and viruses and from noninfectious means such astrauma or reperfusion following myocardial infarction or stroke, immuneresponses to foreign antigens, and autoimmune responses. Accordingly,inflammatory disorders amenable to treatment with a compound or a saltthereof (e.g., a pharmaceutically acceptable salt thereof) of thepresent invention encompass disorders associated with reactions of thespecific defense system as well as with reactions of the nonspecificdefense system.

“Specific defense system” refers to the component of the immune systemthat reacts to the presence of specific antigens. Examples ofinflammation resulting from a response of the specific defense systeminclude the classical response to foreign antigens, autoimmune diseases,and delayed type hypersensitivity responses mediated by T-cells. Chronicinflammatory diseases, the rejection of solid transplanted tissue andorgans, e.g., kidney and bone marrow transplants, and graft versus hostdisease (GVHD), are further examples of inflammatory reactions of thespecific defense system.

The term “nonspecific defense system” refers to inflammatory disordersthat are mediated by leukocytes that are incapable of immunologicalmemory (e.g., granulocytes, and macrophages). Examples of inflammationthat result, at least in part, from a reaction of the nonspecificdefense system include inflammation associated with conditions such asadult (acute) respiratory distress syndrome (ARDS) or multiple organinjury syndromes; reperfusion injury; acute glomerulonephritis; reactivearthritis; dermatoses with acute inflammatory components; acute purulentmeningitis or other central nervous system inflammatory disorders suchas stroke; thermal injury; inflammatory bowel disease; granulocytetransfusion associated syndromes; and cytokine-induced toxicity.

“Autoimmune disease” refers to any group of disorders in which tissueinjury is associated with humoral or cell-mediated responses to thebody's own constituents. Non-limiting examples of autoimmune diseasesinclude rheumatoid arthritis, lupus and multiple sclerosis.

“Allergic disease” as used herein refers to any symptoms, tissue damage,or loss of tissue function resulting from allergy. “Arthritic disease”as used herein refers to any disease that is characterized byinflammatory lesions of the joints attributable to a variety ofetiologies. “Dermatitis” as used herein refers to any of a large familyof diseases of the skin that are characterized by inflammation of theskin attributable to a variety of etiologies. “Transplant rejection” asused herein refers to any immune reaction directed against graftedtissue, such as organs or cells (e.g., bone marrow), characterized by aloss of function of the grafted and surrounding tissues, pain, swelling,leukocytosis, and thrombocytopenia. The therapeutic methods of thepresent invention include methods for the treatment of disordersassociated with inflammatory cell activation.

“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclearleukocytes such as neutrophils, basophils, and eosinophils), mast cells,dendritic cells, Langerhans cells, and endothelial cells). It will beappreciated by persons skilled in the art that the activation of one ora combination of these phenotypes in these cells can contribute to theinitiation, perpetuation, or exacerbation of an inflammatory disorder.

In some embodiments, inflammatory disorders which can be treatedaccording to the methods of this invention include, but are not limitedto, asthma, rhinitis (e.g., allergic rhinitis), allergic airwaysyndrome, atopic dermatitis, bronchitis, rheumatoid arthritis,psoriasis, contact dermatitis, chronic obstructive pulmonary disease(COPD) and delayed hypersensitivity reactions.

The terms “cancer” and “cancerous”, “neoplasm”, and “tumor” and relatedterms refer to or describe the physiological condition in mammals thatis typically characterized by unregulated cell growth. A “tumor”comprises one or more cancerous cells. Examples of cancer includecarcinoma, blastoma, sarcoma, seminoma, glioblastoma, melanoma,leukemia, and myeloid or lymphoid malignancies. More particular examplesof such cancers include squamous cell cancer (e.g., epithelial squamouscell cancer) and lung cancer including small-cell lung cancer, non-smallcell lung cancer (“NSCLC”), adenocarcinoma of the lung and squamouscarcinoma of the lung. Other cancers include skin, keratoacanthoma,follicular carcinoma, hairy cell leukemia, buccal cavity, pharynx(oral), lip, tongue, mouth, salivary gland, esophageal, larynx,hepatocellular, gastric, stomach, gastrointestinal, small intestine,large intestine, pancreatic, cervical, ovarian, liver, bladder,hepatoma, breast, colon, rectal, colorectal, genitourinary, biliarypassage, thyroid, papillary, hepatic, endometrial, uterine, salivarygland, kidney or renal, prostate, testis, vulval, peritoneum, anal,penile, bone, multiple myeloma, B-cell lymphoma, central nervous system,brain, head and neck, Hodgkin's, and associated metastases. Examples ofneoplastic disorders include myeloproliferative disorders, such aspolycythemia vera, essential thrombocytosis, myelofibrosis, such asprimary myelofibrosis, and chronic myelogenous leukemia (CML).

A “chemotherapeutic agent” is an agent useful in the treatment of agiven disorder, for example, cancer or inflammatory disorders. Examplesof chemotherapeutic agents are well-known in the art and includeexamples such as those disclosed in U.S. Publ. Appl. No. 2010/0048557,incorporated herein by reference. Additionally, chemotherapeutic agentsinclude pharmaceutically acceptable salts, acids or derivatives of anyof chemotherapeutic agents, as well as combinations of two or more ofthem.

“Package insert” is used to refer to instructions customarily includedin commercial packages of therapeutic products that contain informationabout the indications, usage, dosage, administration, contraindicationsor warnings concerning the use of such therapeutic products.

Unless otherwise stated, structures depicted herein include compoundsthat differ only in the presence of one or more isotopically enrichedatoms. Exemplary isotopes that can be incorporated into compounds of thepresent invention include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, such as ²H,³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³²F, ³³F, ³⁵S, ¹⁸F, ³⁶Cl,¹²³I, and ¹²⁵I, respectively. Isotopically-labeled compounds (e.g.,those labeled with ³H and ¹⁴C) can be useful in compound or substratetissue distribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e.,¹⁴C) isotopes can be useful for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements). In some embodiments, one or more hydrogenatoms are replaced by ²H or ³H, or one or more carbon atoms are replacedby ¹³C- or ¹⁴C-enriched carbon. Positron emitting isotopes such as ¹⁵O,¹³N, ¹¹C, and ¹⁸F are useful for positron emission tomography (PET)studies to examine substrate receptor occupancy. Isotopically labeledcompounds can generally be prepared by procedures analogous to thosedisclosed in the Schemes or in the Examples herein, by substituting anisotopically labeled reagent for a non-isotopically labeled reagent.

It is specifically contemplated that any limitation discussed withrespect to one embodiment of the invention may apply to any otherembodiment of the invention. Furthermore, any compound or a salt thereof(e.g., a pharmaceutically acceptable salt thereof) or composition of theinvention may be used in any method of the invention, and any method ofthe invention may be used to produce or to utilize any compound or asalt thereof (e.g., a pharmaceutically acceptable salt thereof) orcomposition of the invention.

The use of the term “or” is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

As used herein, “a” or “an” means one or more, unless clearly indicatedotherwise. As used herein, “another” means at least a second or more.

Headings used herein are intended only for organizational purposes.

Inhibitors of Janus Kinases

One embodiment provides a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   R¹ is: C₁₋₆alkyl; cyano-C₁₋₆alkyl; C₁₋₆alkoxy-(CO)—;        —(CHR^(a))_(m)—NR^(b)R^(c); or —(CHR^(a))_(n)-het¹;    -   R² is: C₁₋₆alkyl; hydroxy-C₁₋₆alkyl; halo-C₁₋₆alkyl;        C₁₋₆alkoxy-C₁₋₆alkyl; C₃₋₆cycloalkyl;        —(CHR^(a))_(p)—NR^(b)R^(c); het²; —(CHR^(a))_(q)-het³; or phenyl        which may be unsubstituted or substituted once or twice with        R^(d);    -   R³ is: hydrogen; amino; or C₁₋₆ alkyl;    -   R⁴ is: hydrogen; or C₁₋₆alkyl;    -   R⁵ is: hydrogen; or C₁₋₆alkyl;    -   R⁶ is: hydrogen; C₁₋₆alkyl; or R² and R⁶ together with the atoms        to which they are attached may form a six membered ring;    -   m is from 2 to 3;    -   n is from 0 to 2;    -   p is from 0 to 2;    -   each R^(a) is independently: hydrogen; or C₁₋₆ alkyl;    -   each R^(b) is independently: hydrogen; or C₁₋₆ alkyl;    -   each R^(c) is independently: hydrogen; or C₁₋₆alkyl;    -   het¹ is: tetrahydrofuranyl; azetidinyl; or pyrrolidinyl, each of        which may be unsubstituted or substituted once or twice with        R^(e);    -   het² is: pyridinyl; pyrimidinyl; pyrazolyl; imidazolyl; or        isoquinolinyl which may be partially saturated; each of which        may be unsubstituted or substituted once or twice with R^(f);    -   het³ is: azetidinyl; pyrrolidinyl; oxetanyl; or piperidinyl;        each of which may be unsubstituted or substituted once with        R^(g);    -   each R^(d) is independently: C₁₋₆ alkyl; hydroxy; C₁₋₆        alkoxy-C₁₋₆ alkyl; —(CHR^(a))_(q)—NR^(b)R^(c); or phenyl;    -   each R^(e) is independently: C₁₋₆alkyl; or oxo;    -   each R^(f) is independently: C₁₋₆ alkyl; hydroxy-C₁₋₆ alkyl;        oxo; —(CHR^(a))_(q)—NR^(b)R^(c); —(CHR^(a))_(s)-het⁴;    -   each R^(g) is independently: C₁₋₆ alkyl; or acetyl;    -   q is from 1 to 2;    -   r is from 2 to 3;    -   s is from 2 to 3; and    -   het⁴ is: azetidin-1-yl; 1-methyl-azetidin-3-yl; quinuclidinyl;        1-methyl-pyrrolidin-2-yl; or 4-methylpiperazin-1-yl.

In certain embodiments R³ is hydrogen.

In certain embodiments R⁴ is hydrogen.

In certain embodiments R⁵ is hydrogen.

In certain embodiments R⁶ is hydrogen.

In certain embodiments R¹ is cyanomethyl or methyl.

In certain embodiments R¹ is cyanomethyl.

In certain embodiments R¹ is methyl.

In certain embodiments R² is: C₁₋₆alkyl; hydroxy-C₁₋₆alkyl;halo-C₁₋₆alkyl; C₁₋₆alkoxy-C₁₋₆ alkyl; C₃₋₆ cycloalkyl;—(CHR^(a))_(p)—NR^(b)R^(c); het²; —(CHR^(a))_(q)-het³; or phenyl whichmay be unsubstituted or substituted once with R^(d).

In certain embodiments R² is C₁-6alkyl.

In certain embodiments R² is hydroxy-C₁₋₆ alkyl.

In certain embodiments R² is halo-C₁₋₆ alkyl.

In certain embodiments R² is C₁₋₆ alkoxy-C₁₋₆alkyl.

In certain embodiments R² is C₃₋₆cycloalkyl.

In certain embodiments R² is —(CHR^(a))_(p)—NR^(b)R^(c).

In certain embodiments R² is het².

In certain embodiments R² is —(CHR^(a))_(q)-het³.

In certain embodiments R² is phenyl which may be unsubstituted orsubstituted once with R^(d).

In certain embodiments R² is C₁₋₆ alkyl, hydroxy-C₁₋₆ alkyl;C₁₋₆alkoxy-C₁₋₆ alkyl or halo-C₁₋₆alkyl.

In certain embodiments R² is —(CH₂)₂—NR^(b)R^(c).

In certain embodiments R² is C₁₋₆alkyl.

In certain embodiments R² is hydroxy-C₁₋₆alkyl or C₁₋₆ alkoxy-C₁₋₆alkyl.

In certain embodiments R² is hydroxy-C₁₋₆ alkyl.

In certain embodiments R² is C₁₋₆ alkoxy-C₁₋₆alkyl.

In certain embodiments R² is methoxy-C₁₋₆ alkyl.

In certain embodiments R² is 2-methoxyethyl.

In certain embodiments R² is halo-C₁₋₆ alkyl.

In certain embodiments R² is 2-hydroxyethyl, difluoromethoxy,3-hydroxyhenyl, -methoxyphenyl, 2-hydroxypropyl, pyridine-4-yl, methyl,azetidin-yl, 2-hydroxy-1-methyl-ethyl, methylamino, dimethyl amino,1-methyl-pyrazol-4-yl, phenyl, pyrazol-4-yl, 1-methyl-2-oxo-4-pyridyl,or 2-hydroxy-1-methyl-propyl.

In certain embodiments R² is 2-hydroxyethyl, 2-hydroxypropyl,2-hydroxy-1-methyl-ethyl, or 2-hydroxy-1-methyl-propyl.

In certain embodiments R² is 2-hydroxyethyl.

In certain embodiments R² is 2-hydroxypropyl.

In certain embodiments R² is 2-hydroxy-1-methyl-ethyl.

In certain embodiments R² is 2-hydroxy-1-methyl-propyl.

In certain embodiments m is 2.

In certain embodiments m is 3.

In certain embodiments n is 0.

In certain embodiments n is 1.

In certain embodiments n is 2.

In certain embodiments p is 0.

In certain embodiments p is 1.

In certain embodiments p is 2.

In certain embodiments q is 1.

In certain embodiments q is 2.

In certain embodiments r is 2.

In certain embodiments r is 3.

In certain embodiments s is 2.

In certain embodiments s is 3.

In certain embodiments R^(a) is hydrogen.

In certain embodiments R^(a) is C₁₋₆alkyl.

In certain embodiments R^(b) is hydrogen.

In certain embodiments R^(b) is C₁₋₆alkyl.

In certain embodiments R^(c) is hydrogen.

In certain embodiments R^(c) is C₁₋₆alkyl.

In certain embodiments het′ is 1-methyl azetidin-4-yl,2-oxy-tetrahydrofuran-3-yl or pyrrolidinyl.

In certain embodiments het² is pyridine-4-yl, 1-methyl-pyrazol-4-yl,1-(2-hydroxypropyl)-pyrazol-4-yl, pyrazol-4-yl, pyridine-3-yl,6-oxo-1H-pyridin-3-yl, 1-methyl-2-oxo-pyridin-4-yl,1-quinuclidin-3-ylpyrazol-4-yl,1-[2-[(1-methylpyrrolidin-2-yl]ethyl]pyrazol-4-yl,1-[2-(dimethylamino)propyl]pyrazol-4-yl,1-methyl-azetidin-3-yl)pyrazol-4-yl, 2-methyl-1H-isoquinolin-7-yl,2-methyl-1H-isoquinolin-6-yl, 1-methyl-imidazol-4-yl, or1H-imidazol-4-yl, pyrimidin-5-yl,

In certain embodiments het³ is 1-methyl-azetidin-4-yl, azetidin-4-yl,azetidine-1-yl, pyrrolidin-3-yl, oxetan-4-yl, 1-methyl-pyrrolidin-3-yl,1-acetyl-azetidin-4-yl, piperidin-3-yl, or 4-methylpiperazin-1-yl.

In certain embodiments the subject compounds are of formula (II)

wherein R¹ and R² are as defined herein.

In certain embodiments the subject compounds are of formula (III)

wherein R² is as defined herein.

In certain embodiments the compound of formula (I) is selected from:

or a pharmaceutically acceptable stereoisomer or salt thereof.

In certain embodiments, the subject compound is selected from:

or a stereoisomer or pharmaceutically acceptable salt thereof.

In certain embodiments the compounds are selected from:

or a stereoisomer or pharmaceutically acceptable salt thereof.

In certain embodiments the subject compound is selected from:

Structure

or a stereoisomer or pharmaceutically acceptable salt thereof.

Also provided is a pharmaceutical composition comprising a JAK inhibitoras described herein, or a pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable carrier, dilient or excipient.

Also provided is the use of a JAK inhibitor as described herein, or apharmaceutically acceptable salt thereof in therapy, such as in thetreatment of an inflammatory disease (e.g., asthma). Also provided isthe use of a JAK inhibitor as described herein or a pharmaceuticallyacceptable salt thereof for the preparation of a medicament for thetreatment of an inflammatory disease. Also provided is a method ofpreventing, treating or lessening the severity of a disease or conditionresponsive to the inhibition of a Janus kinase activity in a patient,comprising administering to the patient a therapeutically effectiveamount of a JAK inhibitor as described herein or a pharmaceuticallyacceptable salt thereof.

In one embodiment the disease or condition for therapy is cancer,polycythemia vera, essential thrombocytosis, myelofibrosis, chronicmyelogenous leukemia (CML), rheumatoid arthritis, inflammatory bowelsyndrome, Crohn's disease, psoriasis, contact dermatitis or delayedhypersensitivity reactions.

In one embodiment the use of a JAK inhibitor as described herein or apharmaceutically acceptable salt thereof, for the treatment of cancer,polycythemia vera, essential thrombocytosis, myelofibrosis, chronicmyelogenous leukemia (CML), rheumatoid arthritis, inflammatory bowelsyndrome, Crohn's disease, psoriasis, contact dermatitis or delayedhypersensitivity reactions is provided.

In one embodiment a composition that is formulated for administration byinhalation is provided.

In one embodiment a metered dose inhaler that comprises a compound ofthe present invention or a pharmaceutically acceptable salt thereof isprovided.

In one embodiment a JAK inhibitor as described herein or apharmaceutically acceptable salt thereof is at least five-times morepotent as an inhibitor of JAK1 than as an inhibitor of LRRK2.

In one embodiment a JAK inhibitor as described herein or apharmaceutically acceptable salt thereof is at least ten-times morepotent as an inhibitor of JAK1 than as an inhibitor of LRRK2.

In one embodiment a method for treating hair loss in a mammal comprisingadministering a JAK inhibitor as described herein or a pharmaceuticallyacceptable salt thereof to the mammal is provided.

In one embodiment the use of a JAK inhibitor as described herein or apharmaceutically acceptable salt thereof for the treatment of hair lossis provided.

In one embodiment the use of a JAK inhibitor as described herein or apharmaceutically acceptable salt thereof to prepare a medicament fortreating hair loss in a mammal is provided.

Compounds of the invention may contain one or more asymmetric carbonatoms. Accordingly, the compounds may exist as diastereomers,enantiomers or mixtures thereof. The syntheses of the compounds mayemploy racemates, diastereomers or enantiomers as starting materials oras intermediates. Mixtures of particular diastereomeric compounds may beseparated, or enriched in one or more particular diastereomers, bychromatographic or crystallization methods. Similarly, enantiomericmixtures may be separated, or enantiomerically enriched, using the sametechniques or others known in the art. Each of the asymmetric carbon ornitrogen atoms may be in the R or S configuration and both of theseconfigurations are within the scope of the invention.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the invention. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined. Unless otherwise specified, if solid wedges ordashed lines are used, relative stereochemistry is intended.

Another aspect includes prodrugs of the compounds described herein,including known amino-protecting and carboxy-protecting groups which arereleased, for example hydrolyzed, to yield the compound of the presentinvention under physiologic conditions.

The term “prodrug” refers to a precursor or derivative form of apharmaceutically active substance that is less efficacious to thepatient compared to the parent drug and is capable of beingenzymatically or hydrolytically activated or converted into the moreactive parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy”Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast(1986) and Stella et al., “Prodrugs: A Chemical Approach to TargetedDrug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985). Prodrugs include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, β-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs, and5-fluorocytosine and 5-fluorouridine prodrugs.

A particular class of prodrugs are compounds in which a nitrogen atom inan amino, amidino, aminoalkyleneamino, iminoalkyleneamino or guanidinogroup is substituted with a hydroxy group, an alkylcarbonyl (—CO—R)group, an alkoxycarbonyl (—CO—OR), or an acyloxyalkyl-alkoxycarbonyl(—CO—O—R—O—CO—R) group where R is a monovalent or divalent group, forexample alkyl, alkylene or aryl, or a group having the Formula—C(O)—O-CP1P2-haloalkyl, where P1 and P2 are the same or different andare hydrogen, alkyl, alkoxy, cyano, halogen, alkyl or aryl. In aparticular embodiment, the nitrogen atom is one of the nitrogen atoms ofthe amidino group. Prodrugs may be prepared by reacting a compound withan activated group, such as acyl groups, to bond, for example, anitrogen atom in the compound to the exemplary carbonyl of the activatedacyl group. Examples of activated carbonyl compounds are thosecontaining a leaving group bonded to the carbonyl group, and include,for example, acyl halides, acyl amines, acyl pyridinium salts, acylalkoxides, acyl phenoxides such as p-nitrophenoxy acyl, dinitrophenoxyacyl, fluorophenoxy acyl, and difluorophenoxy acyl. The reactions aregenerally carried out in inert solvents at reduced temperatures such as−78° C. to about 50° C. The reactions may also be carried out in thepresence of an inorganic base, for example potassium carbonate or sodiumbicarbonate, or an organic base such as an amine, including pyridine,trimethylamine, triethylamine, triethanolamine, or the like.

Additional types of prodrugs are also encompassed. For instance, a freecarboxyl group of a JAK inhibitor as described herein can be derivatizedas an amide or alkyl ester. As another example, compounds of the presentinvention comprising free hydroxy groups can be derivatized as prodrugsby converting the hydroxy group into a group such as, but not limitedto, a phosphate ester, hemisuccinate, dimethylaminoacetate, orphosphoryloxymethyloxycarbonyl group, as outlined in Fleisher, D. etal., (1996) Improved oral drug delivery: solubility limitations overcomeby the use of prodrugs Advanced Drug Delivery Reviews, 19:115. Carbamateprodrugs of hydroxy and amino groups are also included, as are carbonateprodrugs, sulfonate esters and sulfate esters of hydroxy groups.Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethylethers, wherein the acyl group can be an alkyl ester optionallysubstituted with groups including, but not limited to, ether, amine andcarboxylic acid functionalities, or where the acyl group is an aminoacid ester as described above, are also encompassed. Prodrugs of thistype are described in J. Med. Chem., (1996), 39:10. More specificexamples include replacement of the hydrogen atom of the alcohol groupwith a group such as (C₁-C₆)alkanoyloxymethyl,1-((C₁-C₆)alkanoyloxy)ethyl, 1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl,(C₁-C₆)alkoxycarbonyloxymethyl, N—(C₁-C₆)alkoxycarbonylaminomethyl,succinoyl, (C₁-C₆)alkanoyl, alpha-amino(C₁-C₄)alkanoyl, arylacyl andalpha-aminoacyl, or alpha-aminoacyl-alpha-aminoacyl, where eachalpha-aminoacyl group is independently selected from the naturallyoccurring L-amino acids, P(O)(OH)₂, —P(O)(O(C₁-C₆)alkyl)₂ or glycosyl(the radical resulting from the removal of a hydroxyl group of thehemiacetal form of a carbohydrate).

“Leaving group” refers to a portion of a first reactant in a chemicalreaction that is displaced from the first reactant in the chemicalreaction. Examples of leaving groups include, but are not limited to,halogen atoms, alkoxy and sulfonyloxy groups. Example sulfonyloxy groupsinclude, but are not limited to, alkylsulfonyloxy groups (for examplemethyl sulfonyloxy (mesylate group) and trifluoromethylsulfonyloxy(triflate group)) and arylsulfonyloxy groups (for examplep-toluenesulfonyloxy (tosylate group) and p-nitrosulfonyloxy (nosylategroup)).

Synthesis of Janus Kinase Inhibitor Compounds

Compounds may be synthesized by synthetic routes described herein. Incertain embodiments, processes well-known in the chemical arts can beused, in addition to, or in light of, the description contained herein.The starting materials are generally available from commercial sourcessuch as Aldrich Chemicals (Milwaukee, Wis.) or are readily preparedusing methods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-19, Wiley, N.Y. (1967-1999 ed.), BeilsteinsHandbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin,including supplements (also available via the Beilstein onlinedatabase)), or Comprehensive Heterocyclic Chemistry, Editors Katrizkyand Rees, Pergamon Press, 1984.

Compounds may be prepared singly or as compound libraries comprising atleast 2, for example 5 to 1,000 compounds, or 10 to 100 compounds.Libraries of compounds may be prepared by a combinatorial ‘split andmix’ approach or by multiple parallel syntheses using either solutionphase or solid phase chemistry, by procedures known to those skilled inthe art. Thus according to a further aspect of the invention there isprovided a compound library comprising at least 2 compounds of thepresent invention.

For illustrative purposes, reaction Schemes depicted below provideroutes for synthesizing the compounds of the present invention as wellas key intermediates. For a more detailed description of the individualreaction steps, see the Examples section below. Those skilled in the artwill appreciate that other synthetic routes may be used. Although somespecific starting materials and reagents are depicted in the Schemes anddiscussed below, other starting materials and reagents can besubstituted to provide a variety of derivatives or reaction conditions.In addition, many of the compounds prepared by the methods describedbelow can be further modified in light of this disclosure usingconventional chemistry well known to those skilled in the art.

In the preparation of compounds of the present invention, protection ofremote functionality (e.g., primary or secondary amine) of intermediatesmay be necessary. The need for such protection will vary depending onthe nature of the remote functionality and the conditions of thepreparation methods. Suitable amino-protecting groups include acetyl,trifluoroacetyl, benzyl, phenylsulfonyl, t-butoxycarbonyl (BOC),benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Theneed for such protection is readily determined by one skilled in theart. For a general description of protecting groups and their use, seeT. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons,New York, 1991.

Other conversions commonly used in the synthesis of compounds of thepresent invention, and which can be carried out using a variety ofreagents and conditions, include the following:

-   (1) Reaction of a carboxylic acid with an amine to form an amide.    Such a transformation can be achieved using various reagents known    to those skilled in the art but a comprehensive review can be found    in Tetrahedron, 2005, 61, 10827-10852.-   (2) Reaction of a primary or secondary amine with an aryl halide or    pseudo halide, e.g., a triflate, commonly known as a    “Buchwald-Hartwig cross-coupling,” can be achieved using a variety    of catalysts, ligands and bases. A review of these methods is    provided in Comprehensive Organic Name Reactions and Reagents, 2010,    575-581.-   (3) A palladium cross-coupling reaction between an aryl halide and a    vinyl boronic acid or boronate ester. This transformation is a type    of “Suzuki-Miyaura cross-coupling,” a class of reaction that has    been thoroughly reviewed in Chemical Reviews, 1995, 95(7),    2457-2483.-   (4) The hydrolysis of an ester to give the corresponding carboxylic    acid is well known to those skilled in the art and conditions    include: for methyl and ethyl esters, the use of a strong aqueous    base such as lithium, sodium or potassium hydroxide or a strong    aqueous mineral acid such as HCl; for a tert-butyl ester, hydrolysis    would be carried out using acid, for example, HCl in dioxane or    trifluoroacetic acid (TFA) in dichloromethane (DCM).-   (5)

Reaction Scheme 1 illustrates a synthesis for compounds of formula I.Nitropyrazole compound 1 can be arylated with4-bromo-1-(difluoromethoxy)-2-iodobenzene under palladium catalyzedconditions to generate compound 2. The nitro group of compound 2 can bereduced with conditions such as iron and ammonium chloride to generateamino aniline 3. Amide bond coupling with commercially availablepyrazolo[1,5-a]pyrimidine-3-carboxylic acid in the presence of acoupling reagent such as, but not limited to, PyAOP, with an organicbase such as, but not limited to DIPEA, and DMAP in an organic solventsuch as, but not limited to, DMF provides compound 4. Removal of the SEMprotecting group of compound 4, using an acid such as, but not limitedto HCl in a solvent such as, but not limited to, 1,4-dioxane, results incompound 5. Compound 5 can then undergo N-alkylation with a group R¹—Xwherein X is halo such as bromo, to provide compound 6. Compound 7 maybe synthesized by treatment of compound 6 with an appropriate thiolreagent R²—SH under Pd catalyzed coupling conditions. Oxidation of thethio group of compound 7 provides compound 8, which is a compound offormula (II) in accordance with the invention. Alternatively, SEMprotected compound 4 can undergo direct N-alkylation by treatment with afluoroborate reagent.

Reaction Scheme 2 illustrates another route to preparation of compoundsof the invention. Nitropyrazyl aryl bromide compound 1 undergoesthiolation in the presence of a palladium catalyist to afford thiolatedcompound 2, after which the nitro group is reduced to the correspondingamine to give compound 3. Amide bond coupling with commerciallyavailable pyrazolo[1,5-a]pyrimidine-3-carboxylic acid in the presence ofa coupling reagent yields compound 4. The SEM protecting group ofcompound 4 is then removed under acidic conditions to give compound 5,which then undergoes S-oxidation to afford the corresponding sulfonecompound 6. N-alkylation of the pyrazole moiety then provides compound(II) in accordance with the invention.

In reaction scheme 3 aryl bromide compound 1 is treated with atrialkylylsilanethiol such as triisopropylsilanethiol to affordthiosilyl compound 2. The thiol is deprotected and alkylated to give thesulfide compound 3, which then may undergo S-oxidation to yield compound(II) in accordance with the invention.

In reaction scheme 4 aryl bromide compound 1 is treated with atrialkylylsilanethiol and undergoes desilylation in situ under basicconditions to provide thiol compound 2. Compound 2 undergoesS-alkylation to provide sulfide compound 3, which in turn is oxidized tocompound II in accordance with the invention.

In certain embodiments, as shown in reaction scheme 5,trialkylsilylthiol compound 1 can undergo oxidation to directly providethe sulfonic acid compound 2, which can then undergo reaction with anamine to provide sulfonamide compound 3.

In certain embodiments, as shown in reaction scheme 6, thiol compound 1is reacted with bromopyrazole to give pyrazole thioester compound 2.Compound 2 is oxidized to yield the corresponding sulfone compound,which may then undergo N-alkylation to afford compound 4.

In reaction scheme 7 sulfone compound 1 is treated with diisopropylazodicarboxylate to give isopropyl carboxylate compound 2.

In reaction scheme 8 aryl bromide compound 1 is reacted with anN-protected thioalkykl amine to give aminoalkyl sulfide compound 2.Compound 2 is oxidized to give the corresponding sulfone, which is thendeprotected to provide aminoalkyl sulfone compound 4. Compound 4 canundergo reductive animation to give compound 5, or alternatively bereacted with acid chloride to give carboxamide compound 6.

In reaction scheme 9 thiol compound 1 is reacted with a substitutedepoxide to give hydroxyalkyl sulfide compound 2. Compound 2 is thenS-oxidized to yield the corresponding hydroxyalkyl sulfone compound 3.

It will be appreciated that where appropriate functional groups exist,compounds of various formulae or any intermediates used in theirpreparation may be further derivatised by one or more standard syntheticmethods employing condensation, substitution, oxidation, reduction, orcleavage reactions. Particular substitution approaches includeconventional alkylation, arylation, heteroarylation, acylation,sulfonylation, halogenation, nitration, formylation and couplingprocedures.

In a further example, primary amine or secondary amine groups may beconverted into amide groups (—NHCOR′ or —NRCOR′) by acylation. Acylationmay be achieved by reaction with an appropriate acid chloride in thepresence of a base, such as triethylamine, in a suitable solvent, suchas dichloromethane, or by reaction with an appropriate carboxylic acidin the presence of a suitable coupling agent such HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) in a suitable solvent such as dichloromethane.Similarly, amine groups may be converted into sulphonamide groups(—NHSO₂R′ or —NR″SO₂R′) groups by reaction with an appropriate sulphonylchloride in the presence of a suitable base, such as triethylamine, in asuitable solvent such as dichloromethane. Primary or secondary aminegroups can be converted into urea groups (—NHCONR′R″ or —NRCONR′R″) byreaction with an appropriate isocyanate in the presence of a suitablebase such as triethylamine, in a suitable solvent, such asdichloromethane.

An amine (—NH₂) may be obtained by reduction of a nitro (—NO₂) group,for example by catalytic hydrogenation, using for example hydrogen inthe presence of a metal catalyst, for example palladium on a supportsuch as carbon in a solvent such as ethyl acetate or an alcohol e.g.,methanol. Alternatively, the transformation may be carried out bychemical reduction using for example a metal, e.g., tin or iron, in thepresence of an acid such as hydrochloric acid.

In a further example, amine (—CH₂NH₂) groups may be obtained byreduction of nitriles (—CN), for example by catalytic hydrogenationusing for example hydrogen in the presence of a metal catalyst, forexample palladium on a support such as carbon, or Raney nickel, in asolvent such as an ether e.g., a cyclic ether such as tetrahydrofuran,at an appropriate temperature, for example from about −78° C. to thereflux temperature of the solvent.

In a further example, amine (—NH₂) groups may be obtained fromcarboxylic acid groups (—CO₂H) by conversion to the corresponding acylazide (—CON₃), Curtius rearrangement and hydrolysis of the resultantisocyanate (—N═C═O).

Aldehyde groups (—CHO) may be converted to amine groups (—CH₂NR′R″)) byreductive amination employing an amine and a borohydride, for examplesodium triacetoxyborohydride or sodium cyanoborohydride, in a solventsuch as a halogenated hydrocarbon, for example dichloromethane, or analcohol such as ethanol, where necessary in the presence of an acid suchas acetic acid at around ambient temperature.

In a further example, aldehyde groups may be converted into alkenylgroups (—CH═CHR′) by the use of a Wittig or Wadsworth-Emmons reactionusing an appropriate phosphorane or phosphonate under standardconditions known to those skilled in the art.

Aldehyde groups may be obtained by reduction of ester groups (such as—CO₂Et) or nitriles (—CN) using diisobutylaluminium hydride in asuitable solvent such as toluene. Alternatively, aldehyde groups may beobtained by the oxidation of alcohol groups using any suitable oxidisingagent known to those skilled in the art.

Ester groups (—CO₂R′) may be converted into the corresponding acid group(—CO₂H) by acid- or base-catalysed hydrolysis, depending on the natureof R. If R is t-butyl, acid-catalysed hydrolysis can be achieved forexample by treatment with an organic acid such as trifluoroacetic acidin an aqueous solvent, or by treatment with an inorganic acid such ashydrochloric acid in an aqueous solvent.

Carboxylic acid groups (—CO₂H) may be converted into amides (CONHR′ or—CONR′R″) by reaction with an appropriate amine in the presence of asuitable coupling agent, such as HATU, in a suitable solvent such asdichloromethane.

In a further example, carboxylic acids may be homologated by one carbon(i.e —CO₂H to —CH₂CO₂H) by conversion to the corresponding acid chloride(—COCl) followed by Arndt-Eistert synthesis.

In a further example, —OH groups may be generated from the correspondingester (e.g., —CO₂R′), or aldehyde (—CHO) by reduction, using for examplea complex metal hydride such as lithium aluminium hydride in diethylether or tetrahydrofuran, or sodium borohydride in a solvent such asmethanol. Alternatively, an alcohol may be prepared by reduction of thecorresponding acid (—CO₂H), using for example lithium aluminium hydridein a solvent such as tetrahydrofuran, or by using borane in a solventsuch as tetrahydrofuran.

Alcohol groups may be converted into leaving groups, such as halogenatoms or sulfonyloxy groups such as an alkylsulfonyloxy, e.g.,trifluoromethylsulfonyloxy or arylsulfonyloxy, e.g.,p-toluenesulfonyloxy group using conditions known to those skilled inthe art. For example, an alcohol may be reacted with thioyl chloride ina halogenated hydrocarbon (e.g., dichloromethane) to yield thecorresponding chloride. A base (e.g., triethylamine) may also be used inthe reaction.

In another example, alcohol, phenol or amide groups may be alkylated bycoupling a phenol or amide with an alcohol in a solvent such astetrahydrofuran in the presence of a phosphine, e.g., triphenylphosphineand an activator such as diethyl-, diisopropyl, ordimethylazodicarboxylate. Alternatively alkylation may be achieved bydeprotonation using a suitable base e.g., sodium hydride followed bysubsequent addition of an alkylating agent, such as an alkyl halide.

Aromatic halogen substituents in the compounds may be subjected tohalogen-metal exchange by treatment with a base, for example a lithiumbase such as n-butyl or t-butyl lithium, optionally at a lowtemperature, e.g., around −78° C., in a solvent such as tetrahydrofuran,and then quenched with an electrophile to introduce a desiredsubstituent. Thus, for example, a formyl group may be introduced byusing N,N-dimethylformamide as the electrophile. Aromatic halogensubstituents may alternatively be subjected to metal (e.g., palladium orcopper) catalysed reactions, to introduce, for example, acid, ester,cyano, amide, aryl, heteraryl, alkenyl, alkynyl, thio- or aminosubstituents. Suitable procedures which may be employed include thosedescribed by Heck, Suzuki, Stille, Buchwald or Hartwig.

Aromatic halogen substituents may also undergo nucleophilic displacementfollowing reaction with an appropriate nucleophile such as an amine oran alcohol. Advantageously, such a reaction may be carried out atelevated temperature in the presence of microwave irradiation.

Methods of Separation

In each of the exemplary Schemes it may be advantageous to separatereaction products from one another or from starting materials. Thedesired products of each step or series of steps is separated orpurified (hereinafter separated) to the desired degree of homogeneity bythe techniques common in the art. Typically such separations involvemultiphase extraction, crystallization or trituration from a solvent orsolvent mixture, distillation, sublimation, or chromatography.Chromatography can involve any number of methods including, for example:reverse-phase and normal phase; size exclusion; ion exchange;supercritical fluid; high, medium, and low pressure liquidchromatography methods and apparatus; small scale analytical; simulatedmoving bed (SMB) and preparative thin or thick layer chromatography, aswell as techniques of small scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved. Example separation methods include boilingpoint, and molecular weight in distillation and sublimation, presence orabsence of polar functional groups in chromatography, stability ofmaterials in acidic and basic media in multiphase extraction, and thelike. One skilled in the art will apply techniques most likely toachieve the desired separation.

Diastereomeric mixtures can be separated into their individualdiastereoisomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereoisomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn or supercritical fluid chromatography.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S., Stereochemistry of OrganicCompounds, John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,J. Chromatogr., 113(3):283-302 (1975)). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: DrugStereochemistry, Analytical Methods and Pharmacology, Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Diastereomeric salts can be formed by reaction of enantiomerically purechiral bases such as brucine, quinine, ephedrine, strychnine,α-methyl-β-phenylethylamine (amphetamine), and the like with asymmetriccompounds bearing acidic functionality, such as carboxylic acid andsulfonic acid. The diastereomeric salts may be induced to separate byfractional crystallization or ionic chromatography. For separation ofthe optical isomers of amino compounds, addition of chiral carboxylic orsulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelicacid, or lactic acid can result in formation of the diastereomericsalts.

Alternatively, the substrate to be resolved is reacted with oneenantiomer of a chiral compound to form a diastereomeric pair (Eliel, E.and Wilen, S., Stereochemistry of Organic Compounds, John Wiley & Sons,Inc., New York, 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob, J. Org. Chem.47:4165 (1982)), of the racemic mixture, and analyzing the NMR spectrumfor the presence of the two atropisomeric enantiomers or diastereomers.Stable diastereomers of atropisomeric compounds can be separated andisolated by normal- and reverse-phase chromatography following methodsfor separation of atropisomeric naphthyl-isoquinolines (WO 96/15111,incorporated herein by reference). By method (3), a racemic mixture oftwo enantiomers can be separated by chromatography using a chiralstationary phase (Chiral Liquid Chromatography W. J. Lough, Ed., Chapmanand Hall, New York, (1989); Okamoto, J. of Chromatogr. 513:375-378(1990)). Enriched or purified enantiomers can be distinguished bymethods used to distinguish other chiral molecules with asymmetriccarbon atoms, such as optical rotation and circular dichroism. Theabsolute stereochemistry of chiral centers and enatiomers can bedetermined by x-ray crystallography.

Positional isomers and intermediates for their synthesis may be observedby characterization methods such as NMR and analytical HPLC. For certaincompounds where the energy barrier for interconversion is sufficientlyhigh, the E and Z isomers may be separated, for example by preparatoryHPLC.

Pharmaceutical Compositions and Administration

The compounds with which the invention is concerned are JAK kinaseinhibitors, such as JAK1 inhibitors, and are useful in the treatment ofseveral diseases, for example, inflammatory diseases, such as asthma.

Accordingly, another embodiment provides pharmaceutical compositions ormedicaments containing a compound of the invention or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier,diluent or excipient, as well as methods of using the compounds of theinvention to prepare such compositions and medicaments.

In one example, a compound of the invention or a pharmaceuticallyacceptable salt thereof may be formulated by mixing at ambienttemperature at the appropriate pH, and at the desired degree of purity,with physiologically acceptable carriers, i.e., carriers that arenon-toxic to recipients at the dosages and concentrations employed intoa galenical administration form. The pH of the formulation dependsmainly on the particular use and the concentration of compound, buttypically ranges anywhere from about 3 to about 8. In one example, acompound of the invention or a pharmaceutically acceptable salt thereofis formulated in an acetate buffer, at pH 5. In another embodiment, thecompounds of the present invention are sterile. The compound may bestored, for example, as a solid or amorphous composition, as alyophilized formulation or as an aqueous solution.

Compositions are formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners.

It will be understood that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination and the severity of the particular diseaseundergoing treatment. Optimum dose levels and frequency of dosing willbe determined by clinical trial, as is required in the pharmaceuticalart. In general, the daily dose range for oral administration will liewithin the range of from about 0.001 mg to about 100 mg per kg bodyweight of a human, often 0.01 mg to about 50 mg per kg, for example 0.1to 10 mg per kg, in single or divided doses. In general, the daily doserange for inhaled administration will lie within the range of from about0.1 μg to about 1 mg per kg body weight of a human, preferably 0.1 μg to50 μg per kg, in single or divided doses. On the other hand, it may benecessary to use dosages outside these limits in some cases.

The compounds of the invention or a pharmaceutically acceptable saltthereof, may be administered by any suitable means, including oral,topical (including buccal and sublingual), rectal, vaginal, transdermal,parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal,intrathecal, inhaled and epidural and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, inhaled administrationis employed.

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, may be administered in any convenient administrative form,e.g., tablets, powders, capsules, lozenges, granules, solutions,dispersions, suspensions, syrups, sprays, vapors, suppositories, gels,emulsions, patches, etc. Such compositions may contain componentsconventional in pharmaceutical preparations, e.g., diluents (e.g.,glucose, lactose or mannitol), carriers, pH modifiers, buffers,sweeteners, bulking agents, stabilizing agents, surfactants, wettingagents, lubricating agents, emulsifiers, suspending agents,preservatives, antioxidants, opaquing agents, glidants, processing aids,colorants, perfuming agents, flavoring agents, other known additives aswell as further active agents.

Suitable carriers and excipients are well known to those skilled in theart and are described in detail in, e.g., Ansel, Howard C., et al.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems.Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R.,et al. Remington: The Science and Practice of Pharmacy. Philadelphia:Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook ofPharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. Forexample, carriers include solvents, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g., antibacterial agents,antifungal agents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, pp 1289-1329, 1990). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.Exemplary excipients include dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof. A pharmaceutical composition maycomprise different types of carriers or excipients depending on whetherit is to be administered in solid, liquid or aerosol form, and whetherit need to be sterile for such routes of administration.

For example, tablets and capsules for oral administration may be in unitdose presentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers, for example, lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example, magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example, potato starch, or acceptable wettingagents such as sodium lauryl sulfate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example, sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example, lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample, almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample, methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavoring or coloring agents.

For topical application to the skin, a compound may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art,for example as described in standard textbooks of pharmaceutics such asthe British Pharmacopoeia.

Compounds of the invention or a pharmaceutically acceptable salt thereofmay also be formulated for inhalation, for example, as a nasal spray, ordry powder or aerosol inhalers. For delivery by inhalation, the compoundis typically in the form of microparticles, which can be prepared by avariety of techniques, including spray-drying, freeze-drying andmicronisation. Aerosol generation can be carried out using, for example,pressure-driven jet atomizers or ultrasonic atomizers, such as by usingpropellant-driven metered aerosols or propellant-free administration ofmicronized compounds from, for example, inhalation capsules or other“dry powder” delivery systems.

By way of example, a composition of the invention may be prepared as asuspension for delivery from a nebulizer or as an aerosol in a liquidpropellant, for example, for use in a pressurized metered dose inhaler(PMDI). Propellants suitable for use in a PMDI are known to the skilledperson, and include CFC-12, HFA-134a, HFA-227, HCFC-22 (CCl₂F₂) andHFA-152 (CH₄F₂ and isobutane).

In some embodiments, a composition of the invention is in dry powderform, for delivery using a dry powder inhaler (DPI). Many types of DPIare known.

Microparticles for delivery by administration may be formulated withexcipients that aid delivery and release. For example, in a dry powderformulation, microparticles may be formulated with large carrierparticles that aid flow from the DPI into the lung. Suitable carrierparticles are known, and include lactose particles; they may have a massmedian aerodynamic diameter of, for example, greater than 90 μm.

In the case of an aerosol-based formulation, an example is:

Compound of the invention* 24 mg/canister

Lecithin, NF Liq. Conc. 1.2 mg/canister

Trichlorofluoromethane, NF 4.025 g/canister

Dichlorodifluoromethane, NF 12.15 g/canister.

* or a pharmaceutically acceptable salt thereof

A compound of the invention or a pharmaceutically acceptable saltthereof may be dosed as described depending on the inhaler system used.In addition to the compound, the administration forms may additionallycontain excipients as described above, or, for example, propellants(e.g., Frigen in the case of metered aerosols), surface-activesubstances, emulsifiers, stabilizers, preservatives, flavorings, fillers(e.g., lactose in the case of powder inhalers) or, if appropriate,further active compounds.

For the purposes of inhalation, a large number of systems are availablewith which aerosols of optimum particle size can be generated andadministered, using an inhalation technique which is appropriate for thepatient. In addition to the use of adaptors (spacers, expanders) andpear-shaped containers (e.g., Nebulator®, Volumatic®), and automaticdevices emitting a puffer spray (Autohaler®), for metered aerosols, inthe case of powder inhalers in particular, a number of technicalsolutions are available (e.g., Diskhaler®, Rotadisk®, Turbohaler® or theinhalers, for example, as described in U.S. Pat. No. 5,263,475,incorporated herein by reference). Additionally, compounds of theinvention or a pharmaceutically acceptable salt thereof, may bedelivered in multi-chamber devices thus allowing for delivery ofcombination agents.

The compound or a pharmaceutically acceptable salt thereof, may also beadministered parenterally in a sterile medium. Depending on the vehicleand concentration used, the compound can either be suspended ordissolved in the vehicle. Advantageously, adjuvants such as a localanaesthetic, preservative or buffering agent can be dissolved in thevehicle.

Targeted Inhaled Drug Delivery

Compounds of the present invention may be used for targeted inhaleddelivery. Optimisation of drugs for delivery to the lung by topical(inhaled) administration has been recently reviewed (Cooper, A. E. etal. Curr. Drug Metab. 2012, 13, 457-473).

Due to limitations in delivery devices, the dose of an inhaled drug maybe limited humans, which necessitates highly potent molecules with goodlung pharmacokinetic properties. High potency against the target ofinterest is especially important for an inhaled drug due to factors suchas the limited amount of drug that can be delivered in a single pufffrom an inhaler, and the safety concerns related to a high aerosolburden in the lung (for example, cough or irritancy). For example, insome embodiments, a Ki of about 0.5 nM or less in a JAK1 biochemicalassay such as described herein, and an IC50 of about 20 nM or less in aJAK1 dependent cell based assay such as described herein, may bedesirable for an inhaled JAK1 inhibitor. In other embodiments, theprojected human dose of a compound of the present invention, or apharmaceutically acceptable salt thereof, is at least two times lessthan the projected human dose of a compound known in the art.Accordingly, in some embodiments, compounds (or a pharmaceuticallyacceptable salt thereof) described herein demonstrate such potencyvalues. The procedures below were used to evaluate the subject compoundsfor potential use as inhaled drugs.

IL13 signaling. IL13 signaling is strongly implicated in asthmapathogenesis. IL13 is a cytokine that requires active JAK1 in order tosignal. Thus, inhibition of JAK1 also inhibits IL13 signaling, which mayprovide benefit to asthma patients. Inhibition of IL13 signaling in ananimal model (e.g., a mouse model) may predict future benefit to humanasthmatic patients. Thus, it may be beneficial for an inhaled JAK1inhibitor to show suppression of IL13 signaling in an animal model.Methods of measuring such suppression are known in the art. For example,as discussed herein and is known in the art, JAK1-dependent STAT6phosphorylation is known to be a downstream consequence of IL13stimulation. Accordingly, in some embodiments, compounds (or apharmaceutically acceptable salt thereof) described herein demonstrateinhibition of lung pSTAT6 induction. To examine pharmacodynamic effectson pSTAT6 levels, compounds of the invention were co-dosed intra-nasallywith IL13 to female Balb/c mice Compounds were formulated in 0.2% (v:v)Tween 80 in saline and mixed 1:1 (v:v) with IL13 immediately prior toadministration. The intranasal doses were administered to lightlyanaesthetised (isoflurane) mice by dispensing a fixed volume (50 μL)directly into the nostrils by pipette to achieve the target dose level(3 mg/kg, 1 mg/kg, 0.3 mg/kg, 0.1 mg/kg). At 0.25 hr post dose, bloodsamples (ca 0.5 mL) were collected by cardiac puncture and plasmagenerated by centrifugation (1500 g, 10 min, +4° C.). The lungs wereperfused with chilled phosphate buffer saline (PBS), weighed and snapfrozen in liquid nitrogen. All samples were stored at ca. −80° C. untilanalysis. Defrosted lung samples were weighed and homogenised followingthe addition of 2 mL HPLC grade water for each gram of tissue, using anOmni-Prep Bead Ruptor at 4° C. Plasma and lung samples were extracted byprotein precipitation with three volumes of acetonitrile containingTolbutamide (50 ng/mL) and Labetalol (25 ng/mL) as analytical internalstandards. Following vortex mixing and centrifugation for 30 minutes at3200 g and 4° C., the supernatants were diluted appropriately (e.g., 1:1v:v) with HPLC grade water in a 96-well plate. Representative aliquotsof plasma and lung samples were assayed for the parent compound byLC-MS/MS, against a series of matrix matched calibration and qualitycontrol standards. The standards were prepared by spiking aliquots ofcontrol Balb/c mouse plasma or lung homogenate (2:1 in HPLC grade water)with test compound and extracting as described for the experimentalsamples. A lung:plasma ratio was determined as the ratio of the meanlung concentration (μM) to the mean plasma concentration (μM) at thesampling time (0.2).

To measure pSTAT6 levels, mouse lungs were stored frozen at −80° C.until assay and homogenised in 0.6 ml ice-cold cell lysis buffer (CellSignalling Technologies, catalogue #9803S) supplemented with 1 mM PMSFand a cocktail of protease (Sigma Aldrich, catalogue #P8340) andphosphatase (Sigma Aldrich, catalogue #P5726 and P0044) inhibitors.Samples were centrifuged at 16060×g for 4 minutes at 4° C. to removetissue debris and protein concentration of homogenates determined usingthe Pierce BCA protein assay kit (catalogue #23225). Samples werediluted to a protein concentration of 5 mg/ml in ice-cold distilledwater and assayed for pSTAT6 levels by Meso Scale Discoveryelectro-chemiluminescent immuno-assay. Briefly, 5 ill/well 150 μg/mlSTAT6 capture antibody (R&D Systems, catalogue #MAB 2169) was coatedonto 96 well Meso Scale Discovery High Binding Plates (catalogue#L15XB-3) and air-dried for 5 hours at room temperature. Plates wereblocked by addition of 1500/well 30 mg/ml Meso Scale Discovery Blocker A(catalogue #R93BA-4) and incubation for 2 hours at room temperature on amicroplate shaker. Blocked plates were washed 4 times with Meso ScaleDiscovery TRIS wash buffer (catalogue #R61TX-1), followed by transfer of50 μl/well lung homogenate to achieve a protein loading of 250 μg/well.Assay plates were incubated overnight at 4° C. and washed 4 times withTRIS wash buffer before addition of 25 μl/well 2.5 μg/mlsulfotag-labelled pSTAT6 detection antibody (BD Pharmingen, catalogue#558241) for 2 hours at room temperature on a microplate shaker. Plateswere washed 4 times with TRIS wash buffer and 150 μl/well 1×Meso ScaleDiscovery Read Buffer T (catalogue #R92TC-1) added. Lung homogenatepSTAT6 levels were quantified by detection of electro-chemiluminescenceon a Meso Scale Discovery SECTOR S 600 instrument.

JAK and JAK2 inhibition Compounds inhibiting both JAK1 and JAK2 arepotentially useful for treatment of different types of asthma.Selectivity between JAK1 and JAK2 may also be important for an inhaledJAK1 inhibitor. For example, GMCSF (granulocyte-macrophagecolony-stimulating factor) is a cytokine that signals through JAK2exclusively. Neutralization of GMCSF activity is associated withpulmonary alveolar proteinosis (PAP) in the lung. However, submaximalJAK2 suppression does not appear to be associated with PAP. Thus, evenmodest JAK1 vs JAK2 selectivity, or approximately equivalent inhibitionof JAK1 and JAK2, may be of benefit in avoiding full suppression of theGMCSF pathway and avoiding PAP. For example, in certain embodimentscompounds that are equipotent for JAK1 and JAK 2 are desirable. In otherembodiments compounds with about 2×-5× selectivity for JAK1 over JAK2may be of benefit for an inhaled JAK1 inhibitor. Accordingly, in someembodiments, compounds (or a pharmaceutically acceptable salt thereof)described herein demonstrate such selectivity. Methods of measuring JAK1and JAK2 selectivity are known in the art, and information can also befound in the Examples herein.

Kinase profiling. Additionally, it may be desirable for an inhaled JAK1or JAK1/JAK2 inhibitor to be selective over one or more other kinases toreduce the likelihood of potential toxicity due to off-target kinasepathway suppression. Thus, it may also be of benefit for an inhaled JAK1inhibitor to be selective against a broad panel of non-JAK kinases, suchas in protocols available from ThermoFisher Scientific's SelectScreen™Biochemical Kinase Profiling Service using Adapta™ Screening ProtocolAssay Conditions (Revised Jul. 29, 2016), LanthaScreen™ Eu KinaseBinding Assay Screening Protocol and Assay Conditions (Revised Jun. 7,2016), and/or Z′LYTE™ Screening Protocol and Assay Conditions (RevisedSep. 16, 2016). For example, a compound of the present invention, or apharmaceutically acceptable salt thereof, exhibits at least 50-foldselectivity for JAK1 versus a panel of non-JAK kinases. Accordingly, insome embodiments, compounds (or a pharmaceutically acceptable saltthereof) described herein demonstrate such selectivity.

Cytotoxicity assays. Hepatocyte toxicity, general cytotoxicity orcytotoxicity of unknown mechanism is an undesirable feature for apotential drug, including inhaled drugs. It may be of benefit for aninhaled JAK1 or JAK1/JAK2 inhibitor to have low intrinsic cytotoxicityagainst various cell types. Typical cell types used to assesscytotoxicity include both primary cells such as human hepatocytes, andproliferating established cell lines such as Jurkat and HEK-293.Accordingly, in some embodiments, compounds (or a pharmaceuticallyacceptable salt thereof) described herein demonstrate such values.Methods of measuring cytotoxicity are known in the art. In someembodiments, compounds described herein were tested as follows:

(a) Jurkat and HEK293T cells were maintained at a sub confluent densityin T175 flasks. Cells were plated at 450 cells/45 μl medium in Greiner384 well black/clear tissue culture treated plates. (Greiner Catalog#781091). After dispensing cells, plates were equilibrated at roomtemperature for 30 minutes. After 30 minutes at room temperature, cellswere incubated overnight at 37° C. in a CO₂ and humidity controlledincubator. The following day, cells were treated with compounds dilutedin 100% DMSO (final DMSO concentration on cells=0.5%) with a 10 pointdose-response curve with a top concentration of 50 μM. Cells andcompounds were then incubated for 72 hours overnight at 37° C. in a CO₂and humidity controlled incubator. After 72 hours of incubation,viability was measured using CellTiterGlo® (Promega Catalog #G7572) toall wells. After incubation at room temperature for 20 minutes, plateswere read on EnVision™ (Perkin Elmer Life Sciences) using luminescencemode;

(b) using human primary hepatocytes: the test compound was prepared as a10 mM solution in DMSO. Additionally, a positive control such asChlorpromazine was prepared as a 10 mM solution in DMSO. Test compoundswere typically assessed using a 7-point dose response curve with 2-folddilutions. Typically, the maximum concentration tested was 50-100 μM.The top concentration was typically dictated by solubility of the testcompound. Cryopreserved primary human hepatocytes(BioreclamationIVT)(lot IZT) were thawed in InVitroGro™ HT thawing media(BioreclamationIVT) at 37° C., pelleted and resuspended. Hepatocyteviability was assessed by Trypan blue exclusion and cells were plated inblack-walled, BioCoat™ collagen 384-well plates (Corning BD) at adensity of 13,000 cells/well in InVitroGro™ CP plating mediasupplemented with 1% Torpedo™ Antibiotic Mix (BioreclamationIVT) and 5%fetal bovine serum. Cells were incubated overnight for 18 hours (37° C.,5% CO₂) prior to treatment. Following 18 hours incubation, plating mediawas removed and hepatocytes were treated with compounds diluted inInVitroGro™ HI incubation media containing 1% Torpedo™ Antibiotic Mixand 1% DMSO (serum-free conditions). Hepatocytes were treated with testcompounds at concentrations such as 0.78, 1.56, 3.12, 6.25, 12.5, 25,and 50 μM at a final volume of 50 μL. A positive control (e.g.,Chlorpromazine) was included in the assay, typically at the sameconcentrations as the test compound. Additional cells were treated with1% DMSO as a vehicle control. All treatments were for a 48 hour timeperiod (at 37° C., 5% CO₂) and each treatment condition was performed intriplicate. Following 48 hours of compound treatment, CellTiter-Glo®cell viability assay (Promega) was used as the endpoint assay to measureATP content as a determination of cell viability. The assay wasperformed according to manufacture instructions. Luminescence wasdetermined on an EnVision™ Muliplate Reader (PerkinElmer, Waltham,Mass., USA). Luminescence data was normalized to vehicle (1% DMSO)control wells. Inhibition curves and IC₅₀ estimates were generated bynon-linear regression of log-transformed inhibitor concentrations(7-point serial dilutions including vehicle) vs. normalized responsewith variable Hill slopes, with top and bottom constrained to constantvalues of 100 and 0, respectively (GraphPad Prism™, GraphPad Software,La Jolla, Calif., USA).

hERG Inhibition. Inhibition of the hERG (human ether-a-go-go-relatedgene) potassium channel may lead to long QT syndrome and cardiacarrhythmias. Although plasma levels of an inhaled JAK1 or JAK1/JAK2inhibitor are expected to be low, lung-deposited compound exiting thelung via pulmonary absorption into the bloodstream will circulatedirectly to the heart. Thus, local heart concentrations of an inhaledJAK1 inhibitor may be transiently higher than total plasma levels,particularly immediately after dosing. Thus, it may be of benefit tominimize hERG inhibition of an inhaled JAK1 inhibitor. For example, insome embodiments, a hERG IC50 greater than 30× over the free-drug plasmaCmax is preferred. Accordingly, in some embodiments, compounds (or apharmaceutically acceptable salt thereof) of the invention demonstrateminimized hERG inhibition under conditions such as:

(a) using hERG 2pt automatic patch clamp conditions to examine in vitroeffects of a compound on hERG expressed in mammalizan cells, evaluatedat room temperature using the QPatch HT® (Sophion Bioscience A/S,Denmark), an automatic parallel patch clamp system. In some cases,compounds were tested at only one or two concentrations such as 1 or 10uM. In other cases a more extensive concentration response relationshipwas established to allow estimation of IC50. For example, test compoundconcentrations were selected to span the range of approximately 10-90%inhibition in half-log increments. Each test article concentration wastested in two or more cells (n>2). The duration of exposure to each testarticle concentration was a minimum of 3 minutes; and/or

(b) those described in WO 2014/074775, in the Examples, under “Effect onCloned hERG Potassium Channels Expressed in Mammalian Cells,” aChanTest™, a Charles River Company, protocol with the following changes:cells stably expressing hERG were held at −80 mV. Onset and steady stateinhibition of hERG potassium current due to compound were measured usinga pulse pattern with fixed amplitudes (conditioning prepulse: +20 mV for1 s; repolarizing test ramepto −90 mV (−0.5 V/s) repeated at 5 sintervals). Each recording ended with a final application of asupramaximal concentration of a reference substance, E-4021 (500 nM)(Charles River Company). The remaining uninhibited current wassubtracted off-line digitially from the data to determine the potency ofthe test substance for hERG inhibition.

CYP (cytochrome P450) inhibition assay. CYP inhibition may not be adesirable feature for an inhaled JAK1 or JAK1/JAK2 inhibitor. Forexample, a reversible or time dependent CYP inhibitor may cause anundesired increase in its own plasma levels, or in the plasma levels ofother co-administered drugs (drug-drug interactions). Additionally, timedependent CYP inhibition is sometimes caused by biotransformation ofparent drug to a reactive metabolite. Such reactive metabolites maycovalently modify proteins, potentially leading to toxicity. Thus,minimizing reversible and time dependent CYP inhibition may be ofbenefit to an inhaled JAK1 inhibitor. Accordingly, in some embodiments,compounds (or a pharmaceutically acceptable salt thereof) of the presentinvention demonstrate minimal or no reversible and/or time dependent CYPinhibition. Methods of measuring CYP inhibition are known in the art.CYP inhibition of compounds described herein were assessed over aconcentration range of 0.16-10 uM of compound using pooled (n=150) humanliver microsomes (Corning, Tewksbury, Mass.) using methods previouslyreported (Halladay et al., Drug Metab. Lett. 2011, 5, 220-230).Incubation duration and protein concentration was dependent on the CYPisoform and the probe substrate/metabolites assessed. The followingsubstrate/metabolites, and incubation times and protein concentrationsfor each CYP were used: CYP1A2, phenacetin/acetaminophen, 30 min, 0.03mg/ml protein; CYP2C9, warfarin/7-hydroxywarfarin, 30 min, 0.2 mg/mlprotein; CYP2C19, mephenytoin/4-hydroxymephenytoin, 40 min, 0.2 mg/mlprotein; CYP2D6, dextromethorphan/dextrorphan, 10 min, 0.03 mg/mlprotein; CYP3A4, midazolam/1-hydroxymidazolam, 10 min, 0.03 mg/mlprotein and CYP3A4 testosterone/6-hydroxytestosterone, 10 min, 0.06mg/ml protein. These conditions were previously determined to be in thelinear rate of formation for the CYP-specific metabolites. All reactionwere initiated with 1 mM NADPH and terminated by the addition of 0.1%formic acid in acetonitrile containing appropriate stable labeledinternal standard. Samples were analyzed by LC-MS/MS

Mouse lung tissue binding. A high bound fraction or percentage ofJAK1/JAK2 inhibitors to lung tissue may be undesirable since it canreduce the amount of free drug available to inhibit JAK1 or JAK2.

(a) Tissue binding experiments were performed in triplicate (n=3) usinga Single-Use RED Plate by following the standard protocol. Initially,individual drugs were spiked to tissue homogenates (pH 7.4) to achieve afinal concentration of 1 μM, and then 300 μL of drug-tissue homogenatemixtures were transferred to the donor wells of the RED plate which waspre-loaded with 500 μL phosphate buffer saline (133 mM) on the receiverwells. The RED plate was sealed with a gas permeable membrane and placedin a shaking incubator (450 rpm, VWR Symphony™) for 6 hr at 37° C. with5% CO₂. At the end of incubation, aliquots of 30 μL samples were takenout of the RED device and matrix equalized with an equal volume oftissue homogenates or buffer, and resulting samples were thenimmediately quenched with ice cold acetonitrile (sample:acetonitrile1:4) containing either propranolol or labetalol as an internal standard.After shaking for 15 min at 500 rpm on a Thermo Scientific CompactDigital MicroPlate Shaker, all samples were then subjected tocentrifugation at 3700 rpm for 15 min (Beckman Coulter Allegra X 12R) toremove plasma protein. Subsequently, supernatants were collected andthen diluted with an equal volume of water prior to LC-MS/MS analysis.

(b) In an alternate procedure, the extent of lung tissue binding of testcompound to mouse lung homogenate may also be determined by equilibriumdialysis using Pierce RED (rapid equilibrium dialysis) devices (FisherScientific 89811 & 89809). A 10 mM solution of compound in DMSO wasprepared and diluted to 1 mM with DMSO. An aliquot of this 1 mM (4 μL)was added into lung homogenate (dilution factor of 1:9, lungtissue:potassium phosphate buffer (0.05 M, pH 7.4)) to give a finalcompound incubation concentration of 5 μM with solvent accounting for0.5% (v/v) of the final incubation volume.

For each assay, the percentage lung tissue bound was determined intriplicate. Lung homogenate (200 μL) was loaded into one side of a REDdevice insert, in triplicate, and 350 μL of potassium phosphate bufferwas loaded into the other side. The RED devices were sealed andincubated for 4 hours at ca. 37° C. on an orbital shaker (−150 rpm).

Following incubation, an aliquot of lung homogenate (8 μL) and analiquot of dialysate (72 μL) were matrix matched (lung homogenate with72 μL phosphate buffer, dialysate with 8 μL lung homogenate) ahead ofanalysis. Protein was precipitated from the samples with the addition of160 μL of acetonitrile containing internal standard. The same matrixmatching and protein precipitation procedure was performed on lunghomogenate aliquots sampled at the start of the experiment (t=0 minsamples), for the assessment of the mass balance. The quenched sampleswere centrifuged (4000 rpm, 30 min, 4° C.) and the resultant supernatantdiluted with water (3:1 (v/v), supernatant: water) and the samplesanalysed for parent compound by liquid chromatography mass spectrometryassay.

The unbound fraction (fu) in lung homogenate was determined from theratio of the dialysate to homogenate peak area, corrected to take intoaccount the lung homogenate dilution (D) to enable an estimate of wholelung tissue binding using the following equations:

Undiluted fu=(1/D)/[((1/Apparent fu)−1)+(1/D)]

Corrected fraction bound (%)=(1−undiluted fu)*100

Kinetic solubility. Good aqueous solubility for JAK1/JAK2 inhibitors forinhaled delivery may be desirable in order to reduce the amount ofundissolved particulate matter in the lung. In one procedure to measurekinetic solubility, 4 μL of a 10 mM DMSO stock solution of test compoundis added to 196 μL of pH 7.4 phosphate buffered saline solution in aMillipore Multiscreen® 96-well filter plate to give a test concentrationof 200 μM with 2% residual DMSO. The filter plate is sealed withaluminum sealing film and shaken at room temperature for 24 hours, thenthe mixtures were vacuum filtered into a clean 96-well plate. Thefiltrate samples are diluted by a factor of two using pH 7.4 phosphatebuffered saline solution, then 5 μL of the resulting solutions areanalyzed by ultra-high performance liquid chromatography (UHPLC) withchemiluminescence nitrogen detection (CLND) and ultraviolet (UV)detection at a wavelength of 254 nm. Sample concentration is typicallyquantified by the CLND intensity, which is related to the number ofnitrogens in the compound. The UV detection is used primarily to confirmsample purity except for rare cases when test compounds contained nonitrogens. In those cases, a compound-specific calibration curve iscollected based on UV absorbance. This curve is then used to determinesample concentration.

Lipophilicity. Lipophilicity is relevant to the solubility, absorption,tissue penetration, protein binding, distribution, and ADME and PKproperties generally of potential drugs. Calculated logP (cLogP), thelogarithm of partition coefficient of a compound between n-octanol andwater (i.e. log(concentration of the compound in n-octanol/concentrationof compound in water), thus can be an important consideration forJAK1/JAK inhibitors for inhaled delivery.

Liver Microsomal Stability. In order to minimize systemic exposure of aninhaled JAK1/JAK2 inhibitor it may be beneficial to optimize for rapidmetabolism in the liver. Liver microsomal stability assay was performedon a BioCel 1200 liquid handling workstation (Agilent Technologies,Santa Clara, Calif.). Compounds (1.0 μM) were incubated for 5 min at 37°C. in 100 μL of a reaction mixture containing 100 mM phosphate buffer(pH 7.4) and 0.5 mg/mL liver microsomes and 1 mM NADPH. At differenttime intervals (0, 20, 40 and 60 min), aliquots of 20 μL of reactionmixtures were taken out and mixed with 4-volumes of acetonitrile (ACN)containing 0.1 μM propranolol as the internal standard to stop metabolicreaction. The samples were then centrifuged at 3250×g for 40 min toremove precipitated protein. The supernatants were subsequentlytransferred to a new 96-well plate and diluted 2-fold using deionizedwater, and were then subjected to LC-MS/MS analysis using an ABI Sciex5500 QTRAP® mass spectrometer (Applied Biosystems, Foster City, Calif.)coupled with a Agilent 1260 HPLC (Agilent Technologies, Santa Clara,Calif.). Percent of remaining was calculated using peak area ratio oftest compound to the internal standard at different time points relativeto the control (T=0 min). See B. Williamson, C. Wilson, G. Dagnell, R JRiley. Harmonised high throughput microsomal stability assay. J.Pharmacol. Toxicol. Methods. 2017; 84:31-36.

Solid state properties. For compounds destined to be delivered via drypowder inhalation there is also a requirement to be able to generatecrystalline forms of the compound that can be micronized to 1-5 μm insize. Particle size is an important determinant of lung deposition of aninhaled compound. Particles with a diameter of less than 5 microns (μm)are typically defined as respirable. Particles with a diameter largerthan 5 μm are more likely to deposit in the oropharynx and arecorrespondingly less likely to be deposited in the lung. Additionally,fine particles with a diameter of less than 1 μm are more likely thanlarger particles to remain suspended in air, and are correspondinglymore likely to be exhaled from the lung. Thus, a particle diameter of1-5 μm may be of benefit for an inhaled medication whose site of actionis in the lung. Typical methods used to measure particle size includelaser diffraction and cascade impaction. Typical values used to defineparticle size include:

-   -   D10, D50, and D90. These are measurements of particle diameter        that indicate, respectively, 10%, 50%, or 90% of the sample is        below that value. For example a D50 of 3 μm indicates that 50%        of the sample is below 3 μm in size.    -   Mass mean aerodynamic diameter (MMAD). MMAD is the diameter at        which 50% of the particles by mass are larger and 50% are        smaller. MMAD is a measure of central tendency.    -   Geometric Standard Deviation (GSD). GSD is a measure of the        magnitude in dispersity from the MMAD, or the spread in        aerodynamic particle size distribution.

A common formulation for inhaled medications is a dry powder preparationincluding the active pharmaceutical ingredient (API) blended with acarrier such as lactose with or without additional additives such asmagnesium stearate. For this formulation and others, it may bebeneficial for the API itself to possess properties that allow it to bemilled to a respirable particle size of 1-5 μm. Agglomeration ofparticles is to be avoided, which can be measured by methods known inthe art, such as examining D90 values under different pressureconditions. Accordingly, in some embodiments, compounds (or apharmaceutically acceptable salt thereof) of the present invention canbe prepared with such a respirable particle size with little or noagglomeration.

As for crystallinity, for some formulations of inhaled drugs, includinglactose blends, it is important that API of a specific crystalline formis used. Crystallinity and crystalline form may impact many parametersrelevant to an inhaled drug including but not limited to: chemical andaerodynamic stability over time, compatibility with inhaled formulationcomponents such as lactose, hygroscopicity, lung retention, and lungirritancy. Thus, a stable, reproducible crystalline form may be ofbenefit for an inhaled drug. Additionally, the techniques used to millcompounds to the desired particle size are often energetic and may causelow melting crystalline forms to convert to other crystalline forms, orto become fully or partially amorphous. A crystalline form with amelting point of less than 150° C. may be incompatible with milling,while a crystalline form with a melting point of less than 100° C. islikely to be non-compatible with milling. Thus, it may be beneficial foran inhaled medication to have a melting point of at least greater than100° C., and ideally greater than 150° C. Accordingly, in someembodiments, compounds (or a pharmaceutically acceptable salt thereof)described herein demonstrate such properties.

Additionally, minimizing molecular weight may help to lower theefficacious dose of an inhaled JAK1 inhibitor. Lower molecular weightresults in a corresponding higher number of molecules per unit mass ofthe active pharmaceutical ingredient (API). Thus, it may be of benefitto find the smallest molecular weight inhaled JAK1 inhibitor thatretains all the other desired properties of an inhaled drug.

Finally, the compound needs to maintain a sufficient concentration inthe lung over a given time period so as to be able to exert apharmacological effect of the desired duration, and for pharmacologicaltargets where systemic inhibition of said target is undesired, to have alow systemic exposure. The lung has an inherently high permeability toboth large molecules (proteins, peptides) as well as small moleculeswith concomitant short lung half-lives, thus it may be necessary toattenuate the lung absorption rate through modification of one or morefeatures of the compounds: minimizing membrane permeability, optimizedpKa, cLogP, solubility, dissolution rate. Methods of measuring suchproperties are known in the art.

Accordingly, in some embodiments, a compound of the present invention(or a pharmaceutically acceptable salt thereof) favourably exhibits oneor more of the above features. Further, in some embodiments, a compoundof the present invention favorably exhibits one or more of thesefeatures relative to a compound known in the art—this may beparticularly true for compounds of the art intended as oral drugs versusinhaled. For example, compounds with rapid oral absorption are typicallypoorly retained in the lung on inhalation.

Methods of Treatment with and Uses of Janus Kinase Inhibitors

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, inhibit the activity of a Janus kinase, such as JAK1,and/or JAK2 kinase. For example, a compound or a pharmaceuticallyacceptable salt thereof inhibits the phosphorylation of signaltransducers and activators of transcription (STATs) by JAK1 kinase aswell as STAT mediated cytokine production. Compounds of the presentinvention are useful for inhibiting JAK1 kinase activity in cellsthrough cytokine pathways, such as IL-6, IL-15, IL-7, IL-2, IL-4, IL-9,IL-10, IL-13, IL-21, G-CSF, IFNalpha, IFNbeta or IFNgamma pathways.Accordingly, in one embodiment is provided a method of contacting a cellwith a compound of the present invention or a pharmaceuticallyacceptable salt thereof, to inhibit a Janus kinase activity in the cell(e.g., JAK1 activity).

The compounds can be used for the treatment of immunological disordersdriven by aberrant IL-6, IL-15, IL-7, IL-2, IL-4, IL9, IL-10, IL-13,IL-21, G-CSF, IFNalpha, IFNbeta or IFNgamma cytokine signaling.

Accordingly, one embodiment includes a compound of the present inventionor a pharmaceutically acceptable salt thereof, for use in therapy.

In some embodiments, there is provided use of a compound of the presentinvention or a pharmaceutically acceptable salt thereof, in thetreatment of an inflammatory disease. Further provided is use of acompound of the present invention or a pharmaceutically acceptable saltthereof for the preparation of a medicament for the treatment of aninflammatory disease, such as asthma. Also provided is a compound of thepresent invention or a pharmaceutically acceptable salt thereof for usein the treatment of an inflammatory disease, such as asthma.

Another embodiment includes a method of preventing, treating orlessening the severity of a disease or condition, such as asthma,responsive to the inhibition of a Janus kinase activity, such as JAK1kinase activity, in a patient. The method can include the step ofadministering to a patient a therapeutically effective amount of acompound of the present invention or a pharmaceutically acceptable saltthereof. In one embodiment, the disease or condition responsive to theinhibition of a Janus kinase, such as JAK1 kinase, is asthma.

In one embodiment, the disease or condition is cancer, stroke, diabetes,hepatomegaly, cardiovascular disease, multiple sclerosis, Alzheimer'sdisease, cystic fibrosis, viral disease, autoimmune diseases,atherosclerosis, restenosis, psoriasis, rheumatoid arthritis,inflammatory bowel disease, asthma, allergic disorders, inflammation,neurological disorders, a hormone-related disease, conditions associatedwith organ transplantation (e.g., transplant rejection),immunodeficiency disorders, destructive bone disorders, proliferativedisorders, infectious diseases, conditions associated with cell death,thrombin-induced platelet aggregation, liver disease, pathologic immuneconditions involving T cell activation, CNS disorders or amyeloproliferative disorder.

In one embodiment, the inflammatory disease is rheumatoid arthritis,psoriasis, asthma, inflammatory bowel disease, contact dermatitis ordelayed hypersensitivity reactions. In one embodiment, the autoimmunedisease is rheumatoid arthritis, lupus or multiple sclerosis.

In another embodiment, a compound of the present invention or apharmaceutically acceptable salt thereof may be used to treat lungdiseases such as a fibrotic lung disease or an interstitial lung disease(e.g., an interstitial pneumonia). In some embodiments, a compound ofthe present invention or a pharmaceutically acceptable salt thereof maybe used to treat idiopathic pulmonary fibrosis (IPF), systemic sclerosisinterstitial lung disease (SSc-ILD)), nonspecific interstitial pneumonia(NSIP), rheumatoid arthritis-associated interstitial lung disease(RA-ILD), sarcoidosis, hypersensitivity pneumonitis, or ILD secondary toconnective tissue disease beyond scleroderma (e.g., polymyositis,dermatomyositis, rheumatoid arthritis, systemic lupus erythematosus(SLE), or mixed connective tissue disease).

In one embodiment, the cancer is breast, ovary, cervix, prostate,testis, penile, genitourinary tract, seminoma, esophagus, larynx,gastric, stomach, gastrointestinal, skin, keratoacanthoma, follicularcarcinoma, melanoma, lung, small cell lung carcinoma, non-small celllung carcinoma (NSCLC), lung adenocarcinoma, squamous carcinoma of thelung, colon, pancreas, thyroid, papillary, bladder, liver, biliarypassage, kidney, bone, myeloid disorders, lymphoid disorders, hairycells, buccal cavity and pharynx (oral), lip, tongue, mouth, salivarygland, pharynx, small intestine, colon, rectum, anal, renal, prostate,vulval, thyroid, large intestine, endometrial, uterine, brain, centralnervous system, cancer of the peritoneum, hepatocellular cancer, headcancer, neck cancer, Hodgkin's or leukemia.

In one embodiment, the disease is a myeloproliferative disorder. In oneembodiment, the myeloproliferative disorder is polycythemia vera,essential thrombocytosis, myelofibrosis or chronic myelogenous leukemia(CML).

Another embodiment includes the use of a compound of the presentinvention or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament for the treatment of a disease describedherein (e.g., an inflammatory disorder, an immunological disorder orcancer). In one embodiment, the invention provides a method of treatinga disease or condition as described herein e.g., an inflammatorydisorder, an immunological disorder or cancer) by targeting inhibitionof a JAK kinase, such as JAK1.

Combination Therapy

The compounds may be employed alone or in combination with other agentsfor treatment. The second or further (e.g., third) compound of apharmaceutical composition or dosing regimen typically has complementaryactivities to the compound of this invention such that they do notadversely affect each other. Such agents are suitably present incombination in amounts that are effective for the purpose intended. Thecompounds may be administered together in a unitary pharmaceuticalcomposition or separately and, when administered separately this mayoccur simultaneously or sequentially. Such sequential administration maybe close or remote in time.

For example, other compounds may be combined with a compound of thepresent invention or a pharmaceutically acceptable salt thereof for theprevention or treatment of inflammatory diseases, such as asthma.Suitable therapeutic agents for a combination therapy include, but arenot limited to: an adenosine A2A receptor antagonist; an anti-infective;a non-steroidal Glucocorticoid Receptor (GR Receptor) agonist; anantioxidant; an alpha 2 adrenoceptor agonist; a CCR1 antagonist; achemokine antagonist (not CCR1); a corticosteroid; a CRTh2 antagonist; aDP1 antagonist; a formyl peptide receptor antagonist; a histonedeacetylase activator; a chloride channel hCLCA1 blocker; an epithelialsodium channel blocker (ENAC blocker; an inter-cellular adhesionmolecule 1 blocker (ICAM blocker); an IKK2 inhibitor; a JNK inhibitor; atransient receptor potential ankyrin 1 (TRPA1) inhibitor; a Bruton'styrosine kinase (BTK) inhibitor (e.g., fenebrutinib); a spleen tyrosinekinase (SYK) inhibitor; a tryptase-beta antibody; an ST2 receptorantibody (e.g., AMG 282); a cyclooxygenase inhibitor (COX inhibitor); alipoxygenase inhibitor; a leukotriene receptor antagonist; a dual alpha2 adrenoceptor agonist/M3 receptor antagonist (MABA compound); a MEK-1inhibitor; a myeloperoxidase inhibitor (MPO inhibitor); a muscarinicantagonist; a p38 MAPK inhibitor; a phosphodiesterase PDE4 inhibitor; aphosphatidylinositol 3-kinase δ inhibitor (PI3-kinase δ inhibitor); aphosphatidylinositol 3-kinase beta inhibitor (PI3-kinase gammainhibitor); a peroxisome proliferator activated receptor agonist(PPARgamma agonist); a protease inhibitor; a retinoic acid receptormodulator (RAR gamma modulator); a statin; a thromboxane antagonist; aTLR7 receptor agonist; or a vasodilator.

In addition, a compound of the present invention or a pharmaceuticallyacceptable salt thereof, may be combined with: (1) corticosteroids, suchas alclometasone dipropionate, amelometasone, beclomethasonedipropionate, budesonide, butixocort propionate, biclesonide, clobetasolpropionate, desisobutyrylciclesonide, dexamethasone, etiprednoldicloacetate, fluocinolone acetonide, fluticasone furoate, fluticasonepropionate, loteprednol etabonate (topical) or mometasone furoate; (2)β2-adrenoreceptor agonists such as salbutamol, albuterol, terbutaline,fenoterol, bitolterol, carbuterol, clenbuterol, pirbuterol, rimoterol,terbutaline, tretoquinol, tulobuterol and long acting β2-adrenoreceptoragonists such as metaproterenol, isoproterenol, isoprenaline,salmeterol, indacaterol, formoterol (including formoterol fumarate),arformoterol, carmoterol, abediterol, vilanterol trifenate, orolodaterol; (3) corticosteroid/long acting β2 agonist combinationproducts such as salmeterol/fluticasone propionate (Advair®, also soldas Seretide®), formoterol/budesonide (Symbicort®),formoterol/fluticasone propionate (Flutiform®), formoterol/ciclesonide,formoterol/mometasone furoate, indacaterol/mometasone furoate,vilanterol trifenate/fluticasone furoate (BREO ELLIPTA), orarformoterol/ciclesonide; (4) anticholinergic agents, for example,muscarinic-3 (M3) receptor antagonists such as ipratropium bromide,tiotropium bromide, aclidinium bromide (LAS-34273), glycopyrroniumbromide, or umeclidinium bromide; (5)M3-anticholinergic/β2-adrenoreceptor agonist combination products suchas vilanterol/umeclidinium (Anoro® Ellipta®), olodaterol/tiotropiumbromide, glycopyrronium bromide/indacaterol (Ultibro®, also sold asXoterna®), fenoterol hydrobromide/ipratropium bromide (Berodual®),albuterol sulfate/ipratropium bromide (Combivent®), formoterolfumarate/glycopyrrolate, or aclidinium bromide/formoterol; (6) dualpharmacology M3-anticholinergic/β2-adrenoreceptor agonists such asbatefenterol succinate, AZD-2115 or LAS-190792; (7) leukotrienemodulators, for example, leukotriene antagonists such as montelukast,zafirulast or pranlukast or leukotriene biosynthesis inhibitors such aszileuton, or LTB4 antagonists such as amelubant, or FLAP inhibitors suchas fiboflapon, GSK-2190915; (8) phosphodiesterase-W (PDE-W) inhibitors(oral or inhaled), such as roflumilast, cilomilast, oglemilast,rolipram, tetomilast, AVE-8112, revamilast, CHF 6001; (9)antihistamines, for example, selective histamine-1 (H1) receptorantagonists such as fexofenadine, citirizine, loratidine or astemizoleor dual H1/H3 receptor antagonists such as GSK 835726, or GSK 1004723;(10) antitussive agents, such as codeine or dextramorphan; (11) amucolytic, for example, N-acetyl cysteine or fudostein; (12) aexpectorant/mucokinetic modulator, for example, ambroxol, hypertonicsolutions (e.g., saline or mannitol) or surfactant; (13) a peptidemucolytic, for example, recombinant human deoxyribonoclease I(dornase-alpha and rhDNase) or helicidin; (14) antibiotics, for exampleazithromycin, tobramycin or aztreonam; (15) non-selective COX-1/COX-2inhibitors, such as ibuprofen or ketoprofen; (16) COX-2 inhibitors, suchas celecoxib and rofecoxib; (17) VLA-4 antagonists, such as thosedescribed in WO 97/03094 and WO 97/02289, each incorporated herein byreference; (18) TACE inhibitors and TNF-α inhibitors, for exampleanti-TNF monoclonal antibodies, such as Remicade® and CDP-870 and TNFreceptor immunoglobulin molecules, such as Enbrel®; (19) inhibitors ofmatrix metalloprotease, for example MMP-12; (20) human neutrophilelastase inhibitors, such as BAY-85-8501 or those described in WO2005/026124, WO 2003/053930 and WO 2006/082412, each incorporated hereinby reference; (21) A2b antagonists such as those described in WO2002/42298, incorporated herein by reference; (22) modulators ofchemokine receptor function, for example antagonists of CCR3 and CCR8;(23) compounds which modulate the action of other prostanoid receptors,for example, a thromboxane A2 antagonist; DP1 antagonists such aslaropiprant or asapiprant CRTH2 antagonists such as 00000459,fevipiprant, ADC 3680 or ARRY 502; (24) PPAR agonists including PPARalpha agonists (such as fenofibrate), PPAR delta agonists, PPAR gammaagonists such as pioglitazone, rosiglitazone and balaglitazone; (25)methylxanthines such as theophylline or aminophylline andmethylxanthine/corticosteroid combinations such astheophylline/budesonide, theophylline/fluticasone propionate,theophylline/ciclesonide, theophylline/mometasone furoate andtheophylline/beclometasone dipropionate; (26) A2a agonists such as thosedescribed in EP1052264 and EP1241176; (27) CXCR2 or IL-8 antagonistssuch as AZD-5069, AZD-4721, or danirixin; (28) IL-R signallingmodulators such as kineret and ACZ 885; (29) MCP-1 antagonists such asABN-912; (30) a p38 MAPK inhibitor such as BCT197, JNJ49095397,losmapimod or PH-797804; (31) TLR7 receptor agonists such as AZD 8848;(32) PI3-kinase inhibitors such as RV1729 or GSK2269557 (nemiralisib);(33) triple combination products such as TRELEGY ELLIPTA (fluticasonefuroate, umeclidinium bromide, and vilanterol); or (34) small moleculeinhibitors of TRPA1, BTK, or SYK.

In some embodiments a compound of the present invention or apharmaceutically acceptable salt thereof, can be used in combinationwith one or more additional drugs, for example anti-hyperproliferative,anti-cancer, cytostatic, cytotoxic, anti-inflammatory orchemotherapeutic agents, such as those agents disclosed in U.S. Publ.Appl. No. 2010/0048557, incorporated herein by reference. A compound ofthe present invention or a pharmaceutically acceptable salt thereof, canbe also used in combination with radiation therapy or surgery, as isknown in the art.

Combinations of any of the foregoing with a compound of the presentinvention or a pharmaceutically acceptable salt thereof are specificallycontemplated.

Articles of Manufacture

Another embodiment includes an article of manufacture (e.g., a kit) fortreating a disease or disorder responsive to the inhibition of a Januskinase, such as a JAK1 kinase. The kit can comprise:

(a) a first pharmaceutical composition comprising a compound of thepresent invention or a pharmaceutically acceptable salt thereof; and

(b) instructions for use.

In another embodiment, the kit further comprises:

(c) a second pharmaceutical composition, such as a pharmaceuticalcomposition comprising an agent for treatment as described above, suchas an agent for treatment of an inflammatory disorder, or achemotherapeutic agent.

In one embodiment, the instructions describe the simultaneous,sequential or separate administration of said first and secondpharmaceutical compositions to a patient in need thereof.

In one embodiment, the first and second compositions are contained inseparate containers. In another embodiment, the first and secondcompositions are contained in the same container.

Containers for use include, for example, bottles, vials, syringes,blister pack, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container includes a compound ofthe present invention or a pharmaceutically acceptable salt thereof,which is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The label or package insert indicates that the compound is usedfor treating the condition of choice, such as asthma or cancer. In oneembodiment, the label or package inserts indicates that the compound canbe used to treat a disorder. In addition, the label or package insertmay indicate that the patient to be treated is one having a disordercharacterized by overactive or irregular Janus kinase activity, such asoveractive or irregular JAK1 activity. The label or package insert mayalso indicate that the compound can be used to treat other disorders.

Alternatively, or additionally, the kit may further comprise a second(or third) container comprising a pharmaceutically acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution or dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

In order to illustrate the invention, the following examples areincluded. However, it is to be understood that these examples do notlimit the invention and are only meant to suggest a method of practicingthe invention. Persons skilled in the art will recognize that thechemical reactions described may be readily adapted to prepare othercompounds of the present invention, and alternative methods forpreparing the compounds are within the scope of this invention. Forexample, the synthesis of non-exemplified compounds according to theinvention may be successfully performed by modifications apparent tothose skilled in the art, e.g., by appropriately protecting interferinggroups, by utilizing other suitable reagents known in the art other thanthose described, or by making routine modifications of reactionconditions. Alternatively, other reactions disclosed herein or known inthe art will be recognized as having applicability for preparing othercompounds of the invention.

EXAMPLES

The following representative compounds of Table 1 were prepared usingprocedures similar to those described in the Schemes and Examplesherein. Absolute stereochemistry of each compound below may not bedepicted: therefore, structures may appear more than once, eachrepresenting a single stereoisomer.

TABLE 1 Exemplary JAK Inhibitors of the Invention Structure Name

N-[3-[2-(difluoromethoxy)-5- isopropylsulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide

N-[3-[5-cyclobutylsulfonyl-2- (difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide

N-[3-[2-(difluoromethoxy)-5-(1- methylazetidin-3-yl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide

N-[3-[5-(azetidin-3-ylsulfonyl)-2- (difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide

N-[3-[2-(difluoromethoxy)-5-(2- hydroxyethylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide

N-[3-[2-(difluoromethoxy)-5- ethylsulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide

N-[3-[2-(difluoromethoxy)-5-[2- (dimethylamino)ethylsulfonyl]phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide

N-[3-[5-(2-aminoethylsulfonyl)-2- (difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide

N-[3-[2-(difluoromethoxy)-5- (difluoromethylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 0

N-[3-[2-(difluoromethoxy)-5- pyrrolidin-3-ylsulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 1

N-[3-[2-(difluoromethoxy)-5-(3-hydroxyphenyl)sulfonyl-phenyl]-1-methyl -pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 2

N-[3-[2-(difluoromethoxy)-5-(3- methoxyphenyl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 3

N-[3-[2-(difluoromethoxy)-5- (oxetan-3-ylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 4

N-[3-[2-(difluoromethoxy)-5-(2- methoxyethylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 5

N-[3-[2-(difluoromethoxy)-5-[rac-(2S)-2-hydroxypropyl]sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 6

N-[3-[2-(difluoromethoxy)-5-(4-pyridylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 7

N-[3-[2-(difluoromethoxy)-5- methylsulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 8

N-[3-[5-[3-(2- aminoethyl)phenyl]sulfonyl-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 9

N-[3-[2-(difluoromethoxy)-5-(1-methylpyrrolidin-3-yl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 0

N-[3-[2-(difluoromethoxy)-5-(1-methylpyrrolidin-3-yl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 1

N-[3-[5-(azetidin-1-ylsulfonyl)-2- (difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 2

N-[3-[5-cyclopropylsulfonyl-2- (difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 3

N-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-ethyl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 4

N-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-ethyl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 5

N-[3-[2-(difluoromethoxy)-5-[rac-(2R)-2-hydroxypropyl]sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 6

N-[3-[2-(difluoromethoxy)-5- (methylsulfamoyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 7

N-[3-[5-(1-acetylazetidin-3- yl)sulfonyl-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 8

N-[1-(cyanomethyl)-3-[2- (difluoromethoxy)-5-methylsulfonyl-phenyl]pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 9

N-[1-(cyanomethyl)-3-[2- (difluoromethoxy)-5-(difluoromethylsulfonyl)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 0

N-[3-[2-(difluoromethoxy)-5- (dimethylsulfamoyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 1

N-[3-[2-(difluoromethoxy)-5- sulfamoyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 2

N-[3-[2-(difluoromethoxy)-5- methylsulfonyl-phenyl]-1-(2-oxotetrahydrofuran-3-yl)pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 3

N-[3-[2-(difluoromethoxy)-5- methylsulfonyl-phenyl]-1-(2-oxotetrahydrofuran-3-yl)pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 4

isopropyl 3-[2-(difluoromethoxy)-5-methylsulfonyl-phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazole-1- carboxylate 5

N-[3-[2-(difluoromethoxy)-5-(1- methylpyrazol-4-yl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 6

N-[3-[2-(difluoromethoxy)-5-(3-piperidylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 7

N-[3-[2-(difluoromethoxy)-5-(3-piperidylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 8

N-[3-[2-(difluoromethoxy)-5-[1- [(2S)-2-hydroxypropyl]pyrazol-4-yl]sulfonyl-phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 9

N-[3-[2-(difluoromethoxy)-5-[2- fluoro-1-(fluoromethyl)ethyl]sulfonyl-phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide0

N-[3-[5-(benzenesulfonyl)-2- (difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 1

N-[3-[2-(difluoromethoxy)-5-(1H- pyrazol-4-ylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 2

N-[3-[2-(difluoromethoxy)-5-(3-pyridylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 3

N-[3-[2-(difluoromethoxy)-5-[(6- oxo-1H-pyridin-3-yl)sulfonyl]phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 4

N-[3-[2-(difluoromethoxy)-5-[(1-methyl-2-oxo-4-pyridyl)sulfonyl]phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 5

N-[1-(cyanomethyl)-3-[5- cyclopropylsulfonyl-2-(difluoromethoxy)phenyl]pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 6

N-[3-[6-(difluoromethoxy)-1,1- dioxo-3,4-dihydro-2H-1lambda6,4-benzothiazin-7-yl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 7

N-[3-[5-[1-(2-aminoethyl)pyrazol-4-yl]sulfonyl-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 8

N-[3-[2-(difluoromethoxy)-5-[1-[2-(methylamino)ethyl]pyrazol-4-yl]sulfonyl- phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 9

N-[3-[2-(difluoromethoxy)-5-[1-[2- (dimethylamino)ethyl]pyrazol-4-yl]sulfonyl-phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 0

N-[3-[5-cyclopropylsulfonyl-2- (difluoromethoxy)phenyl]-1-[2-(dimethylamino)ethyl]pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 1

N-[1-(cyanomethyl)-3-[2- (difluoromethoxy)-5-(oxetan-3-ylsulfonyl)phenyl]pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide2

N-[3-[5-cyclopropylsulfonyl-2- (difluoromethoxy)phenyl]-1-(1-methylazetidin-3-yl)pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 3

N-[3-[5-[1-[2-(azetidin-1- yl)ethyl]pyrazol-4-yl]sulfonyl-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 4

N-[3-[2-(difluoromethoxy)-5-[1-[2-(4-methylpiperazin-1-yl)ethyl]pyrazol-4-yl]sulfonyl-phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 5

N-[3-[2-(difluoromethoxy)-5-(1- quinuclidin-3-ylpyrazol-4-yl)sulfonyl-phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide6

N-[3-[2-(difluoromethoxy)-5-[1-[2- [(2R)-1-methylpyrrolidin-2-yl]ethyl]pyrazol-4-yl]sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 7

N-[3-[2-(difluoromethoxy)-5-[1-[2- [(2S)-1-methylpyrrolidin-2-yl]ethyl]pyrazol-4-yl]sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 8

N-[3-[2-(difluoromethoxy)-5-[1- [(2S)-2-(dimethylamino)propyl]pyrazol-4-yl]sulfonyl-phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 9

N-[3-[2-(difluoromethoxy)-5-[1- [(2R)-2-(dimethylamino)propyl]pyrazol-4-yl]sulfonyl-phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 0

N-[3-[5-cyclopropylsulfonyl-2- (difluoromethoxy)phenyl]-1-[[rac-(2R)-1-methylpyrrolidin-2-yl]methyl]pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 1

N-[3-[2-(difluoromethoxy)-5-[1-(1-methylazetidin-3-yl)pyrazol-4-yl]sulfonyl- phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 2

N-[3-[2-(difluoromethoxy)-5-[(2- methyl-1H-isoquinolin-7-yl)sulfonyl]phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 3

N-[3-[2-(difluoromethoxy)-5-(3- hydroxypropylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 4

N-[3-[2-(difluoromethoxy)-5-[(2- methyl-1H-isoquinolin-6-yl)sulfonyl]phenyl]-1-methyl-pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 5

N-[3-[2-(difluoromethoxy)-5-(1- methylimidazol-4-yl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 6

N-[3-[2-(difluoromethoxy)-5-(1H- imidazol-4-ylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 7

N-[3-[2-(difluoromethoxy)-5- pyrimidin-5-ylsulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3- carboxamide 8

N-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-propyl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 9

N-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-propyl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 0

N-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-propyl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 1

N-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-propyl)sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3-carboxamide 2

N-[3-[2-(difluoromethoxy)-5- methylsulfonyl-phenyl]-1-[[(2S)-1-methylpyrrolidin-2-yl]methyl]pyrazol-4- yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide 3

N-(3-(2-(difluoromethoxy)-5-(N-(2-hydroxyethyl)-N-methylsulfamoyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5- a]pyrimidine-3-carboxamide

LCMS Conditions

Method A

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.2 95  5 2.00 1.2  5 95 2.701.2  5 95 2.75 1.2 95  5 Detection-UV (220 and 254 nm) and ELSD

Method B

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.2 80  20 3.60 1.2 40  60 4.001.2  0 100 4.70 1.2  0 100 4.75 1.2 95  5 Detection-UV (220 and 254 nm)and ELSD

Method C

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.2 95  5 3.00 1.2  5 95 3.701.2  5 95 3.75 1.2 95  5 Detection-UV (220 and 254 nm) and ELSD

Method D

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.2 95  5 3.50 1.2 30  70 3.701.2  0 100 4.50 1.2  0 100 4.75 1.2 95  5 Detection-UV (220 and 254 nm)and ELSD

Method E

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.2 95  5 3.50 1.2 40  60 3.701.2  0 100 4.70 1.2  0 100 4.75 1.2 95  5 Detection-UV (220 and 254 nm)and ELSD

Method F

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.2 70  30 3.50 1.2 30  70 3.701.2  0 100 4.50 1.2  0 100 4.75 1.2 95  5 Detection-UV (220 and 254 nm)and ELSD

Method G

Experiments were performed on a SHIMADZU 20A HPLC with aC18-reverse-phase column (50×2.1 mm Ascentis Express C18, 2.7 μmparticle size), elution with solvent A: water+0.05% trifluoroaceticacid; solvent B: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.0 95  5 1.10 1.0  0 100 1.601.0  0 100 1.70 1.0 95  5 Detection-UV (220 and 254 nm) and ELSD

Method H

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.2 95  5 1.10 1.2  0 100 1.701.2  0 100 1.75 1.2 95  5 Detection-UV (220 and 254 nm) and ELSD

Method I

Experiments were performed on a SHIMADZU 20A HPLC with PoroshellHPH-C18, column (50×3 mm, 2.7 μm particle size), elution with solvent A:water/5 mM NH₄HCO₃; solvent B: acetonitrile. Gradient:

Gradient- flow Time ml/min % A % B 0.00 1.2 90 10 1.10 1.2  5 95 1.601.2  5 95 1.70 1.2 90 10 Detection-UV (220 and 254 nm) and ELSD

Method J

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Kinetex XB-C₁₈, 2.6 μm particle size),elution with solvent A: water+0.05% trifluoroacetic acid; solvent B:acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 1.5 95 5 1.20 1.5 0 100 1.701.5 0 100 1.80 1.5 95 5 Detection - UV (220 and 254 nm) and ELSD

Method K

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 1.0 95 5 2.20 1.0 0 100 3.201.0 0 100 3.30 1.0 95 5 Detection - UV (220 and 254 nm) and ELSD

Method L

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×2.1 mm Kinetex XB-C₁₈ 100A, 2.6 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 1.0 95 5 1.10 1.0 0 100 1.601.0 0 100 1.70 1.0 95 5 Detection - UV (220 and 254 nm) and ELSD

Method M

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (30×2.1 mm Kinetex C18-100A, 1.7 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.01 1.0 95 5 0.60 1.0 0 100 1.001.0 0 100 1.05 1.0 95 5 Detection - UV (220 and 254 nm) and ELSD

Method N

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3.0 mm Poroshell HPH-C18, 2.7 μm particlesize), elution with solvent A: water+5 mM ammonium bicarbonate; solventB: acetonitrile. Gradient:

Gradient - Time flow ml/min % A % B 0.01 1.0 90 10 2.00 1.0 5 95 2.701.0 5 95 2.80 1.0 90 10 Detection - UV (220 and 254 nm) and ELSD

Method O

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3.0 mm Titank C18, 3.0 μm particle size),elution with solvent A: water+5 mM ammonium bicarbonate; solvent B:acetonitrile. Gradient:

Gradient - Time flow ml/min % A % B 0.01 1.0 90 10 2.00 1.0 5 95 2.701.0 5 95 2.80 1.0 90 10 Detection - UV (220 and 254 nm) and ELSD

Method P

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (30×2.1 mm Halo C18, 2.0 μm particle size),elution with solvent A: water+0.05% trifluoroacetic acid; solvent B:acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 1.0 95 5 1.30 1.0 0 100 1.801.0 0 100 1.90 1.0 95 5 Detection - UV (220 and 254 nm) and ELSD

Method Q

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3.0 mm YMC-Triart C18, 2.5 μm particlesize), elution with solvent A: water+0.1% formic acid; solvent B:acetonitrile+0.1% formic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.01 1.0 95 5 3.00 1.0 5 95 3.70 1.05 95 3.75 1.0 95 5 Detection - UV (220 and 254 nm) and ELSD

Method R

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 1.2 70 30 3.10 1.2 0 100 3.701.2 0 100 3.75 1.2 95 5 Detection - UV (220 and 254 nm) and ELSD

Method S

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3.0 mm Poroshell RPH-C18, 2.7 μm particlesize), elution with solvent A: water+5 mM ammonium bicarbonate; solventB: acetonitrile. Gradient:

Gradient - Time flow ml/min % A % B 0.01 1.0 90 10 3.50 1.0 40 60 4.001.0 5 95 4.70 1.0 5 95 4.80 1.0 90 10 Detection - UV (220 and 254 nm)and ELSD

Method T

Experiments were performed on a SHIMADZU 20A HPLC with aC18-reverse-phase column (50×2.1 mm Ascentis Express C18, 2.7 μmparticle size), elution with solvent A: water+0.05% trifluoroaceticacid; solvent B: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 1.0 95 5 2.00 1.0 0 100 2.701.0 0 100 2.80 1.0 95 5 Detection - UV (220 and 254 nm) and ELSD

Method U

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3 mm Shim-Pack XR-ODS, 2.2 μm particlesize), elution with solvent A: water+0.05% trifluoroacetic acid; solventB: acetonitrile+0.05% trifluoroacetic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.01 1.2 95 5 3.50 1.2 50 50 3.701.2 0 100 4.70 1.2 0 100 4.75 1.2 95 5 Detection - UV (220 and 254 nm)and ELSD

Method V

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×3.0 mm Poroshell HPH-C18, 2.7 μm particlesize), elution with solvent A: water+5 mM ammonium bicarbonate; solventB: acetonitrile. Gradient:

Gradient - Time flow ml/min % A % B 0.01 1.0 90 10 3.50 1.0 60 40 4.001.0 5 95 4.70 1.0 5 95 4.80 1.0 90 10 Detection - UV (220 and 254 nm)and ELSD

Method W

Experiments were performed on a SHIMADZU LCMS-2020 with aC18-reverse-phase column (50×2.1 mm Waters Acquity BEH, 1.7 μm particlesize), elution with solvent A: water+0.1% formic acid; solvent B:acetonitrile+0.1% formic acid. Gradient:

Gradient - Time flow ml/min % A % B 0.00 0.8 95 5 1.60 0.8 0 100 1.800.8 0 100 2.00 0.8 95 5 Detection - UV (220 and 254 nm) and ELSD

Method X

Experiments were performed on an Agilent 1290 UHPLC coupled with AgilentMSD (6140) mass spectrometer using ESI as ionization source. The LCseparation was using a Phenomenex XB-C18, 1.7 um, 50×2.1 mm column at aflow rate of 0.4 ml/minute. Mobile phase A was water with 0.1% formicacid and mobile phase B was acetonitrile with 0.1% formic acid. Thegradient started at 2% B and ended at 98% B over 7 min and was held at98% B for 1.5 min following equilibration for 1.5 min. LC columntemperature was 40° C. UV absorbance were collected at 220 nm and 254 nmand mass spec full scan was applied to all experiments.

LIST OF COMMON ABBREVIATIONS

-   ACN Acetonitrile-   Brine Saturated aqueous sodium chloride solution-   CH₃OD Deuterated Methanol-   CDCl₃ Deuterated Chloroform-   DCM Dichloromethane-   DIEA or DIPEA Diisopropylethylamine-   DMA Dimethylacetamide-   DMAP 4-Dimethylaminopyridine-   DMF Dimethylformamide-   DMSO Dimethylsulfoxide-   DMSO-d6 Deuterated dimethylsulfoxide-   DTAD Di-tert-butyl azodicarboxylate-   EDC or EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-   ESI Electrospray ionization-   EtOAc Ethyl acetate-   EtOH Ethanol-   FA Formic Acid-   HOAc Acetic acid-   g Gram-   h hour-   HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate)-   HCl Hydrochloric acid-   HOBt Hydroxybenzotriazole-   HPLC High performance liquid chromatography-   IMS Industrial methylated spirits-   L Liter-   LCMS Liquid chromatography-mass spectrometry-   LiHMDS or LHMDS Lithium hexamethydisylazide-   MDAP Mass directed automated purification-   MeCN Acetonitrile-   MeOH Methanol-   μm Micrometer-   min minute-   mg Milligram-   mL Milliliter-   mm Millimeter-   M Molar-   nm Nanometer-   NMR Nuclear magnetic resonance spectroscopy-   Pd₂(dba)₃.CHCl₃ Tris(dibenzylideneacetone)dipalladium(O)-chloroform    adduct-   PE Petroleum ether-   Prep-HPLC Preparative high performance liquid chromatography-   PyAOP (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium    hexafluorophosphate-   SCX-2 Strong cation exchange-   TBAF Tetra-n-butylammonium fluoride-   THF Tetrahydrofuran-   TFA Trifluoroacetic acid-   Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthine-   ZnCl₂ Zinc chloride

Intermediate 1

N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-02-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis of 4-bromo-1-(difluoromethoxy)-2-iodobenzene

To a solution of 4-bromo-2-iodophenol (282 g, 943 mmol) inN,N-dimethylformamide (2000 mL) and water (500 mL) was added sodium2-chloro-2,2-difluoroacetate (216 g, 1.42 mol) and Cs₂CO₃ (617 g, 1.89mol). The reaction vessel was equipped with a gas outlet for CO₂release. The resulting mixture was stirred overnight at 120° C., allowedto cool to room temperature and poured into ice water (3000 mL). Theresulting solution was extracted with ethyl acetate (3×1500 mL) and theorganic layers were combined. The ethyl acetate extracts were washedwith brine (1000 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel eluting with ethyl acetate/petroleum ether(1/10) to afford 300 g (91%) of4-bromo-1-(difluoromethoxy)-2-iodobenzene as a yellow oil. ¹H NMR (300MHz, CDCl₃) δ 7.96 (dd, J=5.7 Hz, 2.4 Hz, 1H), 7.45 (dd, J=8.7 Hz, 2.4Hz, 1H), 7.03 (d, J=8.7 Hz, 1H), 6.39 (t, J=72.9 Hz, 1H).

Step 2: Synthesis of5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole

To a solution of4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (100 g, 411mmol) in anhydrous THF (1000 mL) was added dropwise to a solution ofLiHMDS (490 mL, 1.0 mol/L in THF) with stirring at −70° C. undernitrogen. The resulting solution was stirred for 1 h at −50° C. and thencooled to −70° C. ZnCl₂ (500 mL, 0.7 mol/L in THF) was added dropwise at−70° C. The resulting solution was allowed to warm to room temperatureand stirred at room temperature for 1 h. To the mixture was added4-bromo-1-(difluoromethoxy)-2-iodobenzene (150 g, 860 mmol), Pd(PPh₃)₄(24.0 g, 20.8 mmol). The resulting solution was heated at refluxtemperature overnight, allowed to cool to room temperature, andconcentrated under reduced pressure. This reaction at this scale wasrepeated one more time, and the crude products from the two runs werecombined for purification. The residue was purified by flashchromatography on silica gel eluting with ethyl acetate/petroleum ether(1/20). The appropriate fractions were combined and concentrated underreduced pressure. This resulted in 300 g (79%) of5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazoleas a light yellow solid in all. ¹H NMR (300 MHz, CDCl₃) δ 8.27 (s, 1H),7.68 (dd, J=8.7, 2.4 Hz, 1H), 7.62 (d, J=2.4 Hz, 1H), 7.19 (d, J=8.4 Hz,1H), 6.39 (t, J=72.5 Hz, 1H), 5.44-5.19 (m, 2H), 3.72-3.54 (m, 2H),0.94-0.89 (m, 2H), 0.02 (s, 9H).

Step 3: Synthesis of5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine

To a solution of5-(5-bromo-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole(50.1 g, 108 mmol) in ethanol (2000 mL) and water (200 mL) was addediron powder (60.1 g, 1.07 mol) and NH₄Cl (28.0 g, 0.523 mol). Thereaction mixture was stirred at reflux temperature for 3 h undernitrogen. The solids were filtered out, and washed with ethanol (100mL). The filtrate was concentrated under reduced pressure. The residuewas dissolved in 3000 mL of ethyl acetate. The ethyl acetate solutionwas washed with 1×500 mL of brine, dried over anhydrous sodium sulfateand concentrated under reduced pressure to give 50.1 g of crude5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amineas a yellow oil. The crude product was used for next step withoutfurther purification. LC/MS (Method G, ESI): [M+H]⁺=434.2, R_(T)=0.93min.

Step 4: Synthesis ofN-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution of5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine(50.1 g, 115 mmol) in DMA (1500 mL) was addedpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (32.1 g, 196.0 mmol), PyAOP(102 g, 196 mmol), DMAP (1.41 g, 11.0 mmol) and DIPEA (44.1 g, 0.341mol). The resulting solution was stirred for 3 h at 60° C. in an oilbath, and then allowed to cool to room temperature. The reaction mixturewas then partitioned between water/ice (2000 mL) and ethyl acetate (2000mL). The aqueous phase was extracted with ethyl acetate (2×). Theorganic layers were combined, washed with brine (1000 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by flash chromatography on silica gel eluting withethyl acetate/petroleum ether (4:1). The appropriate fractions werecombined and concentrated under reduced pressure. Water (150 mL) wasadded to the residue and the mixture was stirred in water for 1 h atroom temperature. The solid was collected by filtration and air-dried toafford 60.1 g (91%) ofN-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideas a light yellow solid. LC/MS (Method G, ESI): [M+H]⁺=579.1 & 581.1,R_(T)=1.10 min. ¹H NMR (300 MHz, CDCl₃) δ 9.62 (s, 1H), 8.80 (dd, J=6.9,1.7 Hz, 1H), 8.73 (s, 1H), 8.53 (dd, J=4.2, 1.7 Hz, 1H), 8.38 (s, 1H),7.79 (d, J=2.4 Hz, 1H), 7.67 (dd, J=8.8, 2.5 Hz, 1H), 7.29 (d, J=1.4 Hz,1H), 7.00 (dd, J=6.9, 4.2 Hz, 1H), 6.43 (t, J=72.6 Hz, 1H), 5.53-5.27(m, 2H), 3.73-3.50 (m, 2H), 0.88 (ddd, J=9.5, 6.4, 4.4 Hz, 2H), 0.00 (s,9H).

N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

N-[5-[5-bromo-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 1, 5.00 g, 8.63 mmol) was treated with HCl/dioxane (150mL, 4 M) overnight at room temperature. The resulting mixture wasconcentrated under reduced pressure. This resulted in 3.80 g ofN-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a yellow solid. The purity of the intermediate was sufficient for usein the next step without further purification. LC/MS (Method I, ESI):[M+H]⁺=449.0, R_(T)=1.02 min. ¹H NMR (400 MHz, CD₃OD) δ 9.11 (dd, J=6.8,1.6 Hz, 1H), 8.67-8.64 (m, 2H), 8.32 (s, 1H), 7.80 (d, J=2.4 Hz, 1H),7.72 (dd, J=8.8, 2.4 Hz, 1H), 7.37 (d, J=8.8 Hz, 1H), 7.23 (dd, J=7.0,4.2 Hz, 1H), 6.81 (t, J=73.2 Hz, 1H).

Intermediate 3

N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[5-[5-bromo-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 1, 10.1 g, 17.3 mmol) in dichloromethane (200 mL) wasadded Me₃OBF₄ (2.81 g, 18.9 mmol) at room temperature. The resultingsolution was stirred for 2 h at room temperature. Then EtOH was added 10mL to the reaction mixture, and the reaction mixture was stirred for 1h, To this solution was added 5.0 mL of HCl (conc.), and it was stirredfor 1 h. The resulting mixture was concentrated under vacuum. The pHvalue of the solution was adjusted to 8 with sodium bicarbonate (20%).The resulting solution was extracted with 3×300 mL of ethyl acetate andthe organic layers combined and dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was applied onto a silica gelcolumn eluting with ethyl acetate/petroleum ether (80%) to give 5.5 g(69%) of N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a lightyellow solid. ¹H NMR (400 MHz, CDCl₃): δ (ppm) 9.86 (s, 1H), 8.80 (dd,J=7.0, 1.6 Hz, 1H), 8.74 (s, 1H), 8.60 (dd, J=4.2, 1.6 Hz, 1H), 8.32 (s,1H), 7.85 (d, J=2.4 Hz, 1H), 7.58 (dd, J=8.4, 2.4 Hz, 1H), 7.24 (d,J=8.8 Hz, 1H), 7.02 (dd, J=7.0, 4.2 Hz, 1H), 6.49 (t, J=74.0 Hz, 1H),4.01 (s, 3H).

Intermediate 4

N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis of5-[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole

Into a 1000-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed toluene (500 mL),5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole(60 g, 129 mmol), NaSMe (26 g, 371 mmol), Pd₂(dba)₃.CHCl₃ (6.7 g, 6.47mmol), XantPhos (7.5 g, 12.96 mmol). The resulting mixture was stirredovernight at 85° C. The resulting mixture was concentrated under vacuum.This reaction was repeated three times. The residue was applied onto asilica gel column eluting with ethyl acetate/petroleum ether (1:20). Theappropriate fractions were combined and concentrated under vacuum. Thisresulted in 171 g of5-[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazoleas a yellow solid in all. LC/MS (Method F, ESI): [M+H]+=432.1, RT=1.23min; ¹H NMR (300 MHz, CDCl₃) δ: (ppm) 8.25 (s, 1H), 7.42 (dd, J=8.7, 2.4Hz, 1H), 7.34 (d, J=2.1 Hz, 1H), 7.23 (d, J=8.7 Hz, 1H), 6.39 (t, J=72.9Hz, 1H), 5.36-5.22 (m, 2H), 3.74-3.55 (m, 2H), 2.51 (s, 3H), 0.94-0.90(m, 2H), 0.02 (s, 9H).

Step 2: Synthesis ofN-[5-[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a mixture of5-[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole(171 g, 408 mmol), ethanol (2000 mL), water (200 mL) was added ironpowder (228 g, 4.08 mol), NH₄Cl (120 g, 2.24 mol). The reaction mixturewas stirred at reflux for 3 h under nitrogen, and cooled to roomtemperature. The solids were filtered out. The filtrate was concentratedunder vacuum. The residue was dissolved in 3000 mL of ethyl acetate andwashed with 1×500 mL of brine. The organic phase was dried overanhydrous sodium sulfate and concentrated under vacuum. This resulted in148 g of5-[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-5-amineas yellow oil. LC/MS (Method F, ESI): [M+H]+=402.1, R_(T)=0.93 min. Intoa 3000-mL 3-necked round-bottom flask, was placed DMA (1500 mL),5[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-amine(148 g), pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (102 g), HATU (325g), 4-dimethylaminopyridine (4.5 g), DIPEA (142 g). The resultingsolution was stirred for 3 h at 60° C., poured into ice water (2000 mL),extracted with 3×2000 mL of ethyl acetate and the organic layerscombined. The resulting mixture was washed with 1×1000 mL of brine. Themixture was dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was applied onto a silica gel column eluting withethyl acetate/petroleum ether (4:1) to give 200 g ofN-[5[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a light yellow solid. LC/MS (Method A, ESI): [M+H]+=547.2, RT=1.10min; ¹H NMR (300 MHz, CDCl₃) δ: (ppm) 9.63 (s, 1H), 8.77 (dd, J=7.0, 1.7Hz, 1H), 8.73 (s, 1H), 8.51 (dd, J=4.2, 1.8 Hz, 1H), 8.38 (s, 1H), 7.50(d, J=2.4 Hz, 1H), 7.39 (dd, J=8.7, 2.4 Hz, 1H), 7.30 (d, J=8.7 Hz, 1H),6.98 (dd, J=6.9, 4.2 Hz, 1H), 6.39 (t, J=73.2 Hz, 1H), 5.46-5.38 (m,2H), 3.70-3.59 (m, 2H), 2.52 (s, 3H), 0.92-0.85 (m, 2H), 0.03 (s, 9H).

Step 3: Synthesis ofN-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[5-[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(60 g) in methanol (600 mL) was added concentrated HCl solution (300mL). The resulting solution was stirred overnight at 35° C. Theresulting mixture was concentrated under vacuum. The solids werecollected by filtration. The solid was suspended in 200 mL of water. ThepH value of the solution was adjusted to 8 with saturated sodiumbicarbonate. The product was collected by filtration, dried to give 30 g(66%)N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideas a light yellow solid. LC/MS (Method G, ESI): [M+H]+=417.0, RT=0.80min; ¹H NMR (300 MHz, DMSO-d₆) δ: (ppm) 13.02 (s, 1H), 9.71 (s, 1H),9.33 (dd, J=6.9, 1.5 Hz, 1H), 8.68 (dd, J=4.1, 1.4 Hz, 1H), 8.66 (s,1H), 8.24 (s, 1H), 7.47-7.36 (m, 3H), 7.27 (dd, J=6.9, 4.2 Hz, 1H), 7.17(t, J=73.8 Hz, 1H), 2.51 (s, 3H).

Intermediate 5

N-(5-(2-(difluoromethoxy)-5-((triisopropylsilyl)thio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 1, 4.00 g, 6.90 mmol) in toluene (20 mL) was added sodiumhydride (415 mg, 10.4 mmol, 60% in mineral oil), Pd₂(dba)₃.CHCl₃ (735mg, 0.710 mmol) and XantPhos (800 mg, 1.38 mmol) andtris(propan-2-yl)silanethiol (1.97 g, 10.4 mmol) under nitrogen. Theresulting solution was stirred for 20 min at 90° C. The resultingmixture was concentrated under vacuum. The residue was purified by flashchromatography on silica gel eluting with ethyl acetate/petroleum ether(80/20). The appropriate fractions were combined and concentrated undervacuum. This resulted in 3.30 g (69%) ofN-(5-(2-(difluoromethoxy)-5-((triisopropylsilyl)thio)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideas an off-white solid. LC/MS (Method B, ESI): [M+H]+=689.4, R_(T)=1.51min.

Intermediate 6

N-[3-[2-(difluoromethoxy)-5-[[tris(propan-2-yl)silyl]sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a suspension of sodium hydride (260 mg, 10.8 mmol) in toluene (40 mL)was added dropwise tris(propan-2-yl)silanethiol (1.23 g, 6.47 mmol) atroom temperature and under nitrogen. The mixture was stirred for 1 h atthis temperature until the mixture turned clear, thenN-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 3, 2.50 g, 5.40 mmol), Pd₂(dba)₃.CHCl₃ (184 mg, 0.178mmol) and XantPhos (250 mg, 0.432 mmol) were added at room temperatureunder nitrogen atmosphere. The resulting solution was stirred for 30 minat 90° C. under N₂. The reaction was then quenched by the addition of 50mL of aqueous NH₄Cl. The resulting solution was diluted with 100 mL ofEA. The organic layer was separated and washed with 3×50 mL of water and2×50 mL of brine. The mixture was dried over anhydrous sodium sulfateand concentrated under vacuum. The residue was applied onto a silica gelcolumn eluting with ethyl acetate/petroleum ether (1/1) to give 2.35 g(76%) of N-[3-[2-(difluoromethoxy)-5-[[tris(propan-2-yl)silyl]sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas an off-white solid. TLC: PE/EA=1/1, Rf=0.4.

Intermediate 7

4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amido]-1H-pyrazol-3-yl)benzene-1-sulfonicacid

Into a 100-mL round-bottom flask, was placedN-[3-[2-(difluoromethoxy)-5-[[tris(propan-2-yl)silyl]sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 6, 360 mg, 0.63 mmol, 1.00 equiv.), dichloromethane (30mL), m-CPBA (217 mg, 1.26 mmol, 2.0 equiv.). The resulting solution wasstirred for 5 h at room temperature. The resulting mixture wasconcentrated under vacuum. The residue was applied onto a silica gelcolumn with dichloromethane/methanol (6:4). This resulted in 210 mg(72%) of4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amido]-1H-pyrazol-3-yl)benzene-1-sulfonicacid as a yellow solid. LC/MS (Method I, ESI): [M+H]+=465.1, R_(T)=0.684min.

Intermediate 8

N-(3-(2-(difluoromethoxy)-5-mercaptophenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 3, 3000 mg, 6.48 mmol), tris(propan-2-yl)silanethiol (1.48g, 1.67 mL, 7.77 mmol), XantPhos Pd G2 (575 mg, 0.648 mmol) and XantPhos(375 mg, 0.648 mmol) in toluene (100 mL) and cooled to 0° C. was addedNaH (518 mg, 13.0 mmol). The reaction mixture was stirred for 30 min at0° C., at room temperature for 1 h, and then stirred at 90° C. for 1 h.The reaction mixture was diluted with dichloromethane, filtered throughCelite, eluting with dichloromethane and the filtrate was concentratedin vacuo. The residue was adsorbed onto silica and purified by flashcolumn chromatography with 10-80% 3:1 MeOH: iPrOAc in Heptane to affordN-(3-(2-(difluoromethoxy)-5-mercaptophenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideas brown solid (2.32 g, 86%). The product was contaminated with somedimer and was used without further purification. LC/MS (Method W, ESI):[M+H]+=417.1, R_(T)=1.09 min.

Intermediate 9

N-(3-(2-(difluoromethoxy)-5-(methylsulfonyl)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 4, 500 mg, 1.20 mmol) in dichloromethane (5.0 mL) wasadded m-CPBA (623 mg, 3.61 mmol). The resulting solution was stirred for5 min at room temperature and concentrated under vacuum. The residue waspurified by flash chromatography on silica gel eluting withdichloromethane/methanol (25/1). The appropriate fractions werecollected and concentrated under vacuum to give 523 mg (97%) ofN-[3-[2-(difluoromethoxy)-5-methanesulfonylphenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a yellow solid. LC/MS (Method G, ESI): [M+H]+=449.2, R_(T)=0.99 min;¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 13.18 (s, 1H), 9.68 (s, 1H), 9.33(dd, J=6.9, 1.5 Hz, 1H), 8.70 (dd, J=4.2, 1.5 Hz, 1H), 8.66 (s, 1H),8.31 (s, 1H), 8.12-8.09 (m, 2H), 7.68 (d, J=8.1 Hz, 1H), 7.47 (t, J=72.6Hz, 1H), 7.30 (dd, 7=6.9, 4.2 Hz, 1H), 3.28 (s, 3H).

EXAMPLES

The following examples are numbered according to the examples in Table 1above.

Example 28

N-(1-(cyanomethyl)-3-(2-(difluoromethoxy)-5-(methylsulfonyl)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 10-mL vial, was placedN-[3-[2-(difluoromethoxy)-5-methanesulfonylphenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 9, 100 mg, 0.223 mmol), cesium carbonate (146 mg, 0.447mmol, 2.00 equiv.), N,N-dimethylformamide (2 mL), and2-bromoacetonitrile (39.8 mg, 0.332 mmol, 1.49 equiv.). The resultingsolution was stirred for 3 h at room temperature. The reaction was thenquenched by the addition of 3 mL of water. The resulting solution wasextracted with 3×3 mL of ethyl acetate and the organic layers combinedand concentrated under vacuum. The crude product (3 mL) was purified byPrep-HPLC with the following conditions (2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, Kinetex EVO C18 Column, 30*150, 5 μm;mobile phase, water (10 mmol/L NH₄HCO₃) and ACN (19.0% ACN up to 27.0%in 7 min); Detector, UV 254/220 nm to afford 40.7 mg (37%) ofN-[1-(cyanomethyl)-3-[2-(difluoromethoxy)-5-methanesulfonylphenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a yellow solid. ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 9.76 (s, 1H), 9.36(dd, J=6.9, 1.5 Hz, 1H), 8.69-8.67 (m, 2H), 8.54 (s, 1H), 8.16 (dd,J=8.7, 2.4 Hz, 1H), 8.08 (d, J=1.6 Hz, 1H), 7.74-7.26 (m, 3H), 5.64 (s,2H), 3.32 (s, 3H). LC/MS (Method A, ESI): [M+H]+=488.2, R_(T)=1.42 min.

Example 22

N-(3-(5-(cyclopropylsulfonyl)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis ofN-[5-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, were placedN-[5-[5-bromo-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 3, 4 g, 6.90 mmol), Pd₂(dba)₃.CHCl₃ (1.4 g, 1.35 mmol,0.20 equiv.), XantPhos (1.6 g, 2.77 mmol, 0.401 equiv.), potassiumcarbonate (1.9 g, 13.7 mmol, 2.0 equiv.), toluene (100 mL), andcyclopropanethiol (2 g, 27 mmol, 3.9 equiv.). The resulting solution wasstirred for 20 h at 100° C. The reaction mixture was cooled to roomtemperature and concentrated under vacuum. The residue was applied ontoa silica gel column with ethyl acetate/hexane (1/1,4/1). The collectedfractions were combined and concentrated under vacuum. This resulted in3.5 g (89%) ofN-[5-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas yellow oil. LC/MS (Method J, ESI): [M+H]+=573.3, R_(T)=1.34 min.

Step 2: Synthesis ofN-[3-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 100-mL round-bottom flask, was placedN-[5-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(3.87 g, 6.76 mmol), dichloromethane (20 mL), and CF₃COOH (10 mL). Theresulting solution was stirred for 4 h at room temperature. Theresulting mixture was concentrated under vacuum. The resulting solutionwas diluted with of ethyl acetate. The solids were collected byfiltration. This resulted in 3.01 g (crude) ofN-[3-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a brown solid. LC/MS (Method H, ESI): [M+H]+=443.15, R_(T)=1.24 min.

Step 3: Synthesis ofN-[3-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 25-mL round-bottom flask, was placedN-[3-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(250 mg, 0.565 mmol), N,N-dimethylformamide (4 mL), cesium carbonate(294 mg, 0.902 mmol, 2.00 equiv.), iodomethane (77 mg, 0.542 mmol, 1.20equiv.). The resulting solution was stirred for 2 h at 30° C. Theresulting mixture was concentrated under vacuum. The residue was appliedonto a silica gel column with ethyl acetate/petroleum ether (2:1). Thisresulted in 150 mg (58%) ofN-[3-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a yellow solid. LC/MS (Method H, ESI): [M+H]+=457.2, R_(T)=1.24 min.

Step 4: Synthesis ofN-[3-[5-(cyclopropanesulfonyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 50-mL round-bottom flask, was placedN-[3-[5-(cyclopropylsulfanyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(280 mg, 0.613 mmol), dichloromethane (20 mL), m-CPBA (212 mg, 1.23mmol, 2.00 equiv.). The resulting solution was stirred for 5 h at roomtemperature. The resulting mixture was concentrated under vacuum. Theresidue was applied onto a silica gel column withdichloromethane/methanol (95:5). The crude product was purified byPrep-HPLC with the following conditions(2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, Kinetex EVO C18 Column,30*150, 5 μm; mobile phase, Water (10 mmol/L NH₄HCO₃) and ACN (25.0% ACNup to 33.0% in 8 min); Detector, UV 254/220 nm. This resulted in 42.9 mg(14%) ofN-3-[5-(cyclopropanesulfonyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.68 (s, 1H), 9.34(dd, J=6.8, 1.4 Hz, 1H), 8.70-8.67 (m, 2H), 8.33 (s, 1H), 8.07 (dd,J=8.8, 2.4 Hz, 1H), 8.02 (d, J=2.4 Hz, 1H), 7.69-7.28 (m, 3H), 3.96 (s,3H), 3.01-2.95 (m, 1H), 1.16-1.12 (m, 2H), 1.08-1.06 (m, 2H). LC/MS(Method A, ESI): [M+H]+=489.2, R_(T)=1.47 min.

Example 52

N-(3-(5-(cyclopropylsulfonyl)-2-(difluoromethoxy)phenyl)-1-(1-methylazetidin-3-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: tert-butyl3-(3-(5-(cyclopropylsulfonyl)-2-(difluoromethoxy)phenyl)-4-(pyrazolo[1,5a]-pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)azetidine-1-carboxylate

To a vial was addedN-[3-[5-cyclopropylsulfonyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(51 mg, 0.11 mmol) and cesium carbonate (105 mg, 0.32 mmol) followed byN,N-dimethylformamide (1.1 mL) andtert-butyl-3-iodoazetidine-1-carboxylate (107 mg, 0.065 mL, 0.38 mmol)and the reaction mixture was heated to 60° C. for 16 h. The reaction wasquenched by the addition of saturated aqueous ammonium chloride andextracted with EtOAc (3×). The combined organic layers were washed withbrine, dried over sodium sulfate, filtered and concentrated in vacuo.The residue was adsorbed onto silica and purified by flash columnchromatography with 10-80% 3:1 MeOH:iPrOAc in heptane to afford thedesired compound as yellow oil (35.6 mg, 53%).

Step 2:N-(1-(azetidin-3-yl)-3-(5-(cyclopropylsulfonyl)-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution of tert-butyl3-[3-[5-cyclopropylsulfonyl-2-(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3-carbonylamino)pyrazol-1-yl]azetidine-1-carboxylate(35.6 mg, 0.057 mmol) in dichloromethane (1 mL) was addedtrifluoroacetic acid (0.25 mL) and the reaction was stirred at roomtemperature for 2 h. The reaction mixture was concentrated in vacuo thentaken up in 2 mL of dichloromethane. MP-carbonate (180 mg, 0.57 mmol)was added and the reaction mixture was stirred for 2 h. The reactionmixture was filtered and the filtrate was concentrated in vacuo toafford the desired compound as a white solid. Assumed full conversionand carried as crude into next step. LC/MS (Method W, ESI):[M+H]+=530.2, R_(T)=0.83 min.

Step 3:N-(3-(5-(cyclopropylsulfonyl)-2-(difluoromethoxy)phenyl)-1-(1-methylazetidin-3-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[1-(azetidin-3-yl)-3-[5-cyclopropylsulfonyl-2-(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(29.9 mg, 0.057 mmol) in 1,2-dichloroethane (1.88 mL) was addedformaldehyde (1.28 mol/L in water) (6.87 mg, 0.0063 mL, 0.085 mmol) andsodium cyanoborohydride (3.62 mg, 0.0031 mL, 0.058 mmol) and thereaction mixture was stirred for 1 h at room temperature. Anotherportion of formaldehyde (1.28 mol/L in water) (6.87 mg, 0.0063 mL, 0.085mmol and sodium cyanoborohydride (3.62 mg, 0.0031 mL, 0.058 mmol) wereadded, and the reaction mixture was stirred overnight. The mixture wasdiluted with dichloromethane and poured into a separatory funnelcontaining saturated aqueous sodium bicarbonate. The layers wereseparated and the aqueous layer was extracted with dichloromethane (2×).The combined organic layers were dried over sodium sulfate, filtered andconcentrated in vacuo. The residue was purified by RP-HPLC to yield thetitle compound (2.1 mg, 7%) as a white solid. LC/MS (Method X, ESI):[M+H]+=544.2, R_(T)=3.06 min.

Example 9

N-(3-(2-(difluoromethoxy)-5-((difluoromethyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1:N-(3-(2-(difluoromethoxy)-5-((difluoromethyl)thio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 50-mL round-bottom flask, was placed sodium2-chloro-2,2-difluoroacetate (617 mg, 4.05 mmol, 1.00 equiv.),N,N-dimethylformamide (10 mL, 129 mmol, 1.00 equiv.), cesium carbonate(704 mg, 2.16 mmol, 2.00 equiv.),N-[3-[2-(difluoromethoxy)-5-[[tris(propan-2-yl)silyl]sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 6, 328 mg, 0.59 mmol, 2.00 equiv.). The resulting solutionwas stirred at 100° C. The resulting mixture was concentrated undervacuum. The residue was applied onto a silica gel column with ethylacetate/petroleum ether (1:1). This resulted in 100 mg (5%) ofN-[3-[2-(difluoromethoxy)-5-[(difluoromethyl)sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a yellow solid. LC/MS (Method H, ESI): [M+H]+=467.2, R_(T)=1.20 min.

Step 2:N-(3-(2-(difluoromethoxy)-5-((difluoromethyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 25-mL round-bottom flask, was placedN-[3-[2-(difluoromethoxy)-5-[(difluoromethyl)sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(100 mg, 0.22 mmol, 1.00 equiv.), dichloromethane (4 mL, 62.9 mmol, 1.00equiv.), m-CPBA (74.4 mg, 0.43 mmol, 2.00 equiv.). The resultingsolution was stirred for 4 h at room temperature. The resulting mixturewas concentrated under vacuum. The residue was applied onto a silica gelcolumn with dichloromethane/methanol (20:1). This resulted in 6.2 mg(6%) ofN-[3-[5-(difluoromethane)sulfonyl-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a yellow solid. ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 9.66 (s, 1H), 9.35(dd, J=6.9, 1.5 Hz, 1H), 8.70-8.67 (m, 2H), 8.35 (s, 1H), 8.18 (dd,J=8.7, 2.4 Hz, 1H), 8.07 (d, J=2.4 Hz, 1H), 7.81-7.22 (m, 4H), 3.97 (s,1H). LC/MS (Method A, ESI): [M+H]+=499.2, R_(T)=1.56 min.

Example 4

N-(3-(5-(azetidin-3-ylsulfonyl)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis of tert-butyl3-((4-(difluoromethoxy)-3-(1-methyl-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazol-3-yl)phenyl)sulfonyl)azetidine-1-carboxylate

Into a 30-mL sealed tube purged and maintained with an inert atmosphereof nitrogen, were placedN-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 3, 1.00 g, 2.16 mmol), Pd₂(dba)₃ (104 mg, 0.114 mmol,0.053 equiv.), tert-butyl 3-sulfanylazetidine-1-carboxylate (500 mg,2.64 mmol, 1.22 equiv.), XantPhos (116 mg, 0.20 mmol, 0.093 equiv.),potassium carbonate (552 mg, 3.99 mmol, 1.85 equiv.), and toluene (15mL). The resulting solution was stirred overnight at 100° C. and thenconcentrated under vacuum. The residue was applied onto a silica gelcolumn with dichloromethane/methanol (94/6). The collected fractionswere combined and concentrated under vacuum. This resulted in 1.5 g(122%) of tert-butyl3-[[4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amido]-1H-pyrazol-3-yl)phenyl]sulfanyl]azetidine-1-carboxylateas a red solid. LC/MS (Method A, ESI): [M+H]+=572.3, R_(T)=1.28 min.

Step 2: Synthesis of tert-butyl3-[[4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amido]-1H-pyrazol-3-yl)benzene]sulfonyl]azetidine-1-carboxylate

Into a 50-mL round-bottom flask, were placed tert-butyl3-[[4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amido]-1H-pyrazol-3-yl)phenyl]sulfanyl]azetidine-1-carboxylate(100 mg, 0.175 mmol), dichloromethane (10 mL), and3-chlorobenzene-1-carboperoxoic acid (60 mg, 0.35 mmol, 2.0 equiv.). Theresulting solution was stirred for 2 h at room temperature. Theresulting mixture was concentrated under vacuum. The residue was appliedonto a silica gel column with dichloromethane/methanol (92/8). Thecollected fractions were combined and concentrated under vacuum. Thisresulted in 100 mg (95%) of tert-butyl3-[[4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amido]-1H-pyrazol-3-yl)benzene]sulfonyl]azetidine-1-carboxylateas a brown solid. LC/MS (Method I, ESI): [M+H]+=604.3, R_(T)=1.05 min.

Step 3: Synthesis ofN-(3-(5-(azetidin-3-ylsulfonyl)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 50-mL round-bottom flask, were placed tert-butyl3-[[4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amido]-1H-pyrazol-3-yl)benzene]sulfonyl]azetidine-1-carboxylate(100 mg, 0.166 mmol, 1.00 equiv.), trifluoroacetic acid (1 mL, 13.5mmol, 82 equiv.), and dichloromethane (10 mL). The resulting solutionwas stirred for 6 h at room temperature. The resulting mixture wasconcentrated under vacuum. The crude product (5 mL) was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,silica gel; mobile phase, H₂O (NH₄HCO₃)/CH₃CN=90/10 increasing toH₂O(NH₄HCO₃)/CH₃CN=50/50 within 10 min; Detector, UV 254 nm. Thisresulted in 11.6 mg (14%) ofN-[3-[5-(azetidine-3-sulfonyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.67 (s, 1H),9.35 (dd, J=6.8, 1.6 Hz, 1H), 8.70-8.66 (m, 2H), 8.34 (s, 1H), 8.07 (dd,J=4.4, 1.6 Hz, 1H), 8.00 (s, J=2.4 Hz, 1H), 7.70-7.29 (m, 3H), 4.67-4.59(m, 1H), 3.97 (s, 3H), 3.80-3.77 (m, 2H), 3.60-3.56 (m, 2H). LC/MS(Method A, ESI): [M+H]+=504.2, R_(T)=1.12 min.

Example 3

N-(3-(2-(difluoromethoxy)-5-((1-methylazetidin-3-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 50-mL round-bottom flask, were placedN-[3-[5-(azetidine-3-sulfonyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(50 mg, 0.099 mmol, 1.00 equiv.), methanol (1 mL), NaBH(AcO)₃ (200 mg,0.94 mmol, 9.5 equiv.), and dichloromethane (10 mL). The resultingsolution was stirred overnight at room temperature. The resultingmixture was concentrated under vacuum. The crude product (5 mL) waspurified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, silica gel; mobile phase,H₂O(NH₄HCO₃)/CH₃CN=90/10 increasing to H₂O(NH₄HCO₃)/CH₃CN=50/50 within10 min; Detector, UV 254 nm. This resulted in 18.1 mg (35%) ofN-[3-[2-(difluoromethoxy)-5-(1-methylazetidine-3-sulfonyl)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.67 (s, 1H),9.35 (dd, J=6.8, 1.6 Hz, 1H), 8.70-8.66 (m, 2H), 8.34 (s, 1H), 8.07 (dd,J=4.4, 1.6 Hz, 1H), 8.00 (s, J=2.4 Hz, 1H), 7.70-7.29 (m, 3H), 4.67-4.59(m, 1H), 3.97 (s, 3H), 3.80-3.77 (m, 2H), 3.60-3.56 (m, 2H). LC/MS(Method A, ESI): [M+H]+=518.3, R_(T)=1.14 min.

N-(3-(54(1-acetylazetidin-3-yl)sulfonyl)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 50-mL round-bottom flask, was placedN-[3-[5-(azetidine-3-sulfonyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(80 mg, 0.159 mmol), dichloromethane (15 mL), pyridine (60 mg, 0.76mmol, 4.8 equiv.). Acetyl chloride (15 mg, 0.19 mmol, 1.20 equiv.) wasadded into the solution at 0° C. The resulting solution was stirred for30 min at room temperature. The reaction was then quenched by theaddition of 10 mL of water. The resulting solution was extracted with2×20 mL of dichloromethane and the organic layers combined, dried, andconcentrated under vacuum. The residue was applied onto a silica gelcolumn with dichloromethane/methanol (93:7). The crude product waspurified by Prep-HPLC with the following conditions(2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, Kinetex EVO C18 Column,30*150, 5 μm; mobile phase, Water (10 mmol/L NH₄HCO₃) and ACN (21.0% ACNup to 25.0% in 8 min); Detector, UV 254/220 nm. This resulted in 5.8 mg(7%) ofN-[3-[5-(1-acetylazetidine-3-sulfonyl)-2-(difluoromethoxy)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.68 (s, 1H), 9.36(dd, J=6.8, 1.6 Hz, 1H), 8.70-8.68 (m, 2H), 8.34 (s, 1H), 8.16 (dd,J=6.8, 2.4 Hz, 1H), 8.07 (d, J=2.4 Hz, 1H), 7.72-7.30 (m, 3H), 4.62-4.55(m, 1H), 4.38-4.34 (m, 2H), 4.07-3.98 (m, 2H), 3.97 (s, 3H), 2.08 (s,3H). LC/MS (Method A, ESI): [M+H]+=546.2, R_(T)=1.32 min.

Example 26

N-[3-[2-(difluoromethoxy)-5-(methylsulfamoyl)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 25-mL 2-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, were placed4-(difluoromethoxy)-3-(1-methyl-4-[pyrazolo[1,5-a]pyrimidine-3-amido]-1H-pyrazol-3-yl)benzene-1-sulfonicacid (Intermediate 7, 150 mg, 0.323 mmol), dichloromethane (10 mL),N,N-dimethylformamide (0.05 mL, 0.646 mmol, 2.00 equiv.), oxalylchloride (0.50 mL, 5.87 mmol, 18.2 equiv.), pyridine (100 mg, 1.26 mmol,3.91 equiv.), and methylamine (0.50 mL, 14.4 mmol, 44.7 equiv.). Theresulting solution was stirred for 1 h at room temperature. The reactionwas then quenched by the addition of 10 mL of water. The resultingsolution was extracted with 2×20 mL of dichloromethane and the organiclayers combined and dried over anhydrous sodium sulfate and concentratedunder vacuum. The residue was applied onto a silica gel column withdichloromethane/methanol (93:7). The crude product (20 mg) was purifiedby Prep-HPLC with the following conditions(2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, Kinetex EVO C18 Column,30*150, 5 μm; mobile phase, Water (10 MMOL/L NH₄HCO₃) and ACN (20% ACNup to 35% in 7 min); Detector, UV 254/220 nm. This resulted in 8.6 mg(6%) ofN-[3-[2-(difluoromethoxy)-5-(methylsulfamoyl)phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.70 (s, 1H), 9.35(dd, J=7.2, 1.6 Hz, 1H), 8.70-8.68 (m, 2H), 8.34 (s, 1H), 7.96-7.93 (m,2H), 7.66-7.26 (m, 4H), 3.96 (s, 3H), 2.44 (s, 3H). LC/MS (Method A,ESI): [M+H]+=478.2, R_(T)=1.39 min.

Example 5

N-(3-(2-(difluoromethoxy)-5-((2-hydroxyethyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis ofN-(3-(2-(difluoromethoxy)-5-((2-hydroxyethyl)thio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 3, 20.0 g, 43.2 mmol), DIEA (11.2 g, 86.5 mmol),Pd₂(dba)₃.CH₃Cl (2.24 g, 2.16 mmol) and XantPhos (2.5 g, 4.32 mmol) in1,4-dioxane (200 mL) and under nitrogen, was added 2-mercaptoethanol(3.19 mL, 45.3 mmol) at room temperature. The resulting solution wasstirred for 4 h at 95° C. The resulting mixture was cooled to roomtemperature and filtered. The filtrate was concentrated under vacuum.The crude residue was purified by flash chromatography on silica geleluting with MeOH/DCM (3%) to affordN-(3-(2-(difluoromethoxy)-5-((2-hydroxyethyl)thio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide(16.8 g, 36.5 mmol, 84.5% yield) as an off-white solid. LC/MS (Method A,ESI): [M+H]+=461.1, R_(T)=1.02 min.

Step 2: Synthesis ofN-[3-[2-(difluoromethoxy)-5-(2-hydroxyethylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

Potassium peroxomonosulfate (125 g, 204 mmol) was dissolved in water(450 mL) at 25° C. while stirring and then added into a suspension ofN-(3-(2-(difluoromethoxy)-5-((2-hydroxyethyl)thio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide(45 g, 97 mmol) in methanol (900 mL). The resulting suspension wasstirred over 6 h at room temperature. Water (1 L) was added, and theresulting suspension was concentrated under vaccum to remove methanol.After filtration, the solids were collected and washed with water (100mL). The solid was dried under 50° C. and slurried with water (500 mL).After filtration, the solids were collected and washed with water (50mL). The solid was dried at 50° C. overnight to afford 44 g. The solidwas slurried with EtOH (400 mL) at 80° C. over 3 h and then cooled downto room temperature in oil bath. After filtration, the solids werecollected and washed by EtOH (20 mL). The solid was slurried with EtOH(300 mL) at 80° C. over 3 h and then cooled down to room temperature inoil bath and stirred overnight. After filtration, the solids werecollected and washed by EtOH (50 mL). The solid was dried under 50° C.overnight to affordN-[3-[2-(difluoromethoxy)-5-(2-hydroxyethylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(43.3 g, 87.9 mmol, 90.4% yield) as an off-white solid. ¹H NMR (400 MHz,DMSO-d6) 9.68 (s, 1H), 9.34 (dd, J=7.0, 1.6 Hz, 1H), 8.68 (dd, J=4.2,1.6 Hz, 1H), 8.66 (s, 1H), 8.33 (s, 1H), 8.11 8.01 (m, 2H), 7.67-7.29(m, 3H), 4.89 (t, J=5.3 Hz, 1H), 3.96 (s, 3H), 3.71 (m, 2H), 3.53 (t,J=6.2 Hz, 2H). LC/MS (Method A, ESI): [M+H]+=493.2, R_(T)=1.27 min.

Example 34

Isopropyl3-(2-(difluoromethoxy)-5-(methylsulfonyl)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazole-1-carboxylate

Into a 100-mL 3-necked round-bottom flask, were placedN-[3-[2-(difluoromethoxy)-5-methanesulfonylphenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(100 mg, 0.223 mmol), triphenylphosphine (76 mg, 0.29 mmol, 1.3 equiv.),DIAD (58.6 mg, 0.290 mmol, 1.30 equiv.), and toluene (6 mL). Theresulting solution was stirred overnight at 95° C. The resulting mixturewas concentrated under vacuum. The residue was applied onto a silica gelcolumn with ethyl acetate/petroleum ether (4:1). This resulted in 20 mg(17%) of isopropyl3-(2-(difluoromethoxy)-5-(methylsulfonyl)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1H-pyrazole-1-carboxylateas a white solid. ¹H NMR (300 MHz, DMSO-d6) 9.87 (s, 1H), 9.37 (dd,J=7.2, 1.5 Hz, 1H), 8.78 (s, 1H), 8.71 (s, 1H), 8.64 (dd, J=4.2, 1.8 Hz,1H), 8.25 (dd, J=8.9, 2.7 Hz, 1H), 8.16 (d, J=2.4 Hz, 1H), 7.80-7.26 (m,3H), 5.26-5.18 (m, 1H), 3.33 (s, 1H), 1.42 (d, J=6.3 Hz, 1H). LC/MS(Method A, ESI): [M+H]+=535.2, R_(T)=1.68 min.

Example 12

N-(3-(2-(difluoromethoxy)-5-((3-methoxyphenyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis ofN-[3-[2-(difluoromethoxy)-5-[(3-methoxyphenyl)sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 30-mL sealed tube purged and maintained with an inert atmosphereof nitrogen, was placedN-[3-[2-(difluoromethoxy)-5-[[tris(propan-2-yl)silyl]sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 6, 1.10 g, 1.92 mmol, 1.00 equiv.), cesium fluoride (658mg, 4.33 mmol, 2.26 equiv.), isopropanol (20 mL),1-bromo-3-methoxybenzene (809 mg, 4.33 mmol, 2.25 equiv.),Pd₂(dba)₃.CHCl₃ (117 mg, 0.113 mmol, 0.059 equiv.), cesium carbonate(1.40 g, 4.30 mmol, 2.24 equiv.). The resulting solution was stirred for6 h at 90° C. The resulting mixture was concentrated under vacuum. Theresidue was applied onto a silica gel column withdichloromethane/methanol (95/5). The collected fractions were combinedand concentrated under vacuum. This resulted in 1.2 g (120%) ofN-[3-[2-(difluoromethoxy)-5-[(3-methoxyphenyl)sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas a red solid.

Step 2: Synthesis ofN-(3-(2-(difluoromethoxy)-5-((3-methoxyphenyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 50-mL round-bottom flask, was placedN-[3-[2-(difluoromethoxy)-5-[(3-methoxyphenyl)sulfanyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(800 mg, 1.53 mmol), dichloromethane (10 mL),3-chlorobenzene-1-carboperoxoic acid (753 mg, 4.36 mmol, 1.00 equiv.).The resulting solution was stirred for 2 h at room temperature. Themixture was concentrated and the crude product was purified by columnchromatography to affordN-(3-(2-(difluoromethoxy)-5-((3-methoxyphenyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide.¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 9.67 (s, 1H), 9.32 (dd, 1.5 Hz, 1H),8.65-8.60 (m, 2H), 8.31 (s, 1H), 8.20 (dd, J=8.7, 2.4 Hz, 1H), 8.08-7.20(m, 7H), 3.95 (s, 3H), 3.81 (s, 3H). LC/MS (Method A, ESI):[M+H]+=555.3, R_(T)=1.65 min.

Example 11

N-(3-(2-(difluoromethoxy)-5-((3-hydroxyphenyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placedN-[3-[2-(difluoromethoxy)-5-[(3-methoxybenzene)sulfonyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(410 mg, 0.739 mmol), dichloromethane (10 mL), and tribromoborane (1.5mL, 12.0 mmol, 16.2 equiv.). The resulting solution was stirred for 3 hat room temperature. The resulting mixture was concentrated under vacuumand purified by column chromatography. This resulted in 76.5 mg (19%) ofN-[3-[2-(difluoromethoxy)-5-[(3-hydroxybenzene)sulfonyl]phenyl]-1-methyl-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas an off-white solid. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.26 (s, 1H),9.67 (s, 1H), 9.32 (dd, J=7.2, 1.5 Hz, 1H), 8.66-8.63 (m, 2H), 8.33 (s,1H), 8.12 (dd, J=8.8, 2.4 Hz, 1H), 8.02 (d, J=2.4 Hz, 1H), 7.66-7.26 (m,6H), 7.06-7.01 (m, 1H), 3.96 (s, 3H). LC/MS (Method A, ESI):[M+H]+=541.3, R_(T)=1.51 min.

Example 35

N-(3-(2-(difluoromethoxy)-5-((1-methyl-1H-pyrazol-4-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis ofN-[3-[2-(difluoromethoxy)-5-(1-methylpyrazol-4-yl)sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a vial were addedN-[3-[2-(difluoromethoxy)-5-sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 8, 1.05 g, 2.52 mmol) and XantPhos Pd G3 (201 mg, 0.202mmol). N,N-dimethylformamide (16.8 mL) was added followed by4-bromo-1-methyl-1H-pyrazole (837 mg, 0.537 mL, 5.04 mmol) andN,N-diisopropylethylamine (978 mg, 1.32 mL, 7.56 mmol), and nitrogen wasbubbled through the reaction mixture for 3 min. The reaction mixture washeated to 180° C. in a microwave reactor for 30 min. The reactionmixture was quenched by the addition of saturated aqueous ammoniumchloride and extracted with ethyl acetate (3×). The combined organiclayers were washed with brine, dried over sodium sulfate, filtered andconcentrated in vacuo. The residue was adsorbed onto silica and purifiedby flash column chromatography with 10-80% 3:1 MeOH: iPrOAc in heptaneto affordN-[3-[2-(difluoromethoxy)-5-(1-methylpyrazol-4-yl)sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas yellow foam (555 mg, 44%). LC/MS (Method W, ESI): [M+H]+=497.2R_(T)=1.09 min.

Step 2: Synthesis ofN-(3-(2-(difluoromethoxy)-5-((1-methyl-1H-pyrazol-4-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-(1-methylpyrazol-4-yl)sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(651 mg, 1.31 mmol) in dichloromethane (16.4 mL) was added3-chloro-perbenzoic acid (1.18 g, 5.25 mmol) and the reaction wasstirred at room temperature for 30 min. The reaction mixture wasconcentrated in vacuo and the residue was dissolved in ethyl acetate andwashed with saturated aqueous ammonium chloride. The aqueous layer wasextracted with EtOAc (2×), and the combined organic layers were washedwith brine, dried over sodium sulfate, filtered and concentrated invacuo. This reaction was repeated scaling toN-[3-[2-(difluoromethoxy)-5-(1-methylpyrazol-4-yl)sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(555 mg), and the two portions were combined for recrystallization. Thecombined lots were fully dissolved in 410 mL of boiling EtOH. Heatingwas stopped and the solution was allowed to cool to room temperatureslowly, with stirring, then cooled in an ice bath. The precipitate wasfiltered, collected and dried under vacuum to affordN-(3-(2-(difluoromethoxy)-5-((1-methyl-1H-pyrazol-4-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide(372 mg, 29%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.64 (s, 1H), 9.33 (dd,J=7.0, 1.7 Hz, 1H), 8.67-8.61 (m, 2H), 8.51 (s, 1H), 8.32 (s, 1H), 8.10(dd, J=8.8, 2.5 Hz, 1H), 8.03 (d, J=2.5 Hz, 1H), 7.97 (d, J=0.9 Hz, 1H),7.64 (d, J=8.8 Hz, 1H), 7.42 (t, J=72.5 Hz, 1H), 7.28 (dd, J=7.0, 4.3Hz, 1H), 3.95 (s, 3H), 3.83 (s, 3H). LC/MS (Method X, ESI): [M+H]+=529.1R_(T)=3.96 min.

Example 41

N-(3-(5-((1H-pyrazol-4-yl)sulfonyl)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis ofN-[3-[2-(difluoromethoxy)-5-(1H-pyrazol-4-ylsulfanyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a vial was addedN-[3-[2-(difluoromethoxy)-5-sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 8, 1000 mg, 2.40 mmol), 4-bromo-1H-pyrazole (706 mg, 4.80mmol), and XantPhos Pd G3 (240 mg, 0.240 mmol). N,N-Dimethylformamide(16.0 mL) was added followed by N,N-diisopropylethylamine (931 mg, 1.26mL, 7.20 mmol), and nitrogen was bubbled through the reaction mixturefor 3 min. The reaction mixture was heated to 180° C. in a microwavereactor for 45 min. The reaction mixture was quenched by the addition ofsaturated aqueous ammonium chloride and extracted with EtOAc (3×). Thecombined organic layers were washed with brine, dried over sodiumsulfate, filtered and concentrated in vacuo. The residue was adsorbedonto silica and purified by flash column chromatography with 10-80% 3:1MeOH: iPrOAc in heptane to affordN-[3-[2-(difluoromethoxy)-5-(1H-pyrazol-4-ylsulfanyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamideas an orange solid (517 mg, 45%).

Step 2: Synthesis ofN-(3-(5-((1H-pyrazol-4-yl)sulfonyl)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-(1H-pyrazol-4-ylsulfanyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(517 mg, 1.07 mmol) in dichloromethane (10 mL) was added3-chloroperbenzoic acid (961 mg, 4.29 mmol) and the reaction was stirredat room temperature for 30 min. The reaction mixture was diluted withdichloromethane and poured into a separatory funnel containing saturatedaqueous sodium bicarbonate. The layers were separated and the aqueouslayer was extracted with dichloromethane (2×). The combined organiclayers were dried over sodium sulfate, filtered and concentrated invacuo. The residue was adsorbed onto silica and purified by flash columnchromatography with 10-80% 3:1 MeOH:iPrOAc in heptane to affordN-(3-(5-((1H-pyrazol-4-yl)sulfonyl)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideas brown solid (383 mg, 69%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.78 (s, 1H),9.64 (s, 1H), 9.32 (dd, J=7.0, 1.7 Hz, 1H), 8.65 (s, 1H), 8.63 (dd,J=4.3, 1.7 Hz, 1H), 8.40-8.18 (m, 3H), 8.11 (dd, J=8.8, 2.5 Hz, 1H),8.05 (d, J=2.5 Hz, 1H), 7.63 (d, J=8.7 Hz, 1H), 7.41 (t, J=72.5 Hz, 1H),7.27 (dd, J=7.0, 4.2 Hz, 1H), 3.95 (s, 3H). LC/MS (Method X, ESI):[M+H]+=515.1 R_(T)=3.71 min.

Example 53

N-(3-(5-((1-(2-(azetidin-1-yl)ethyl)-1H-pyrazol-4-yl)sulfonyl)-2-(difluoromethoxy)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-(1H-pyrazol-4-ylsulfonyl)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(50 mg, 0.097 mmol), PS-triphenylphosphine (85.0 mg, 0.20 mmol),di-tert-butyl azodicarboxylate (49.2 mg, 0.21 mmol) in tetrahydrofuran(1.5 mL) was added 2-(azetidin-1-yl)ethanol (20.3 mg, 0.019 mL, 0.19mmol) and the reaction mixture was heated to 60° C. for 90 min. Thereaction mixture was filtered and the filtrate was concentrated invacuo. The crude mixture was taken up in dichloromethane (3 mL) and HCl(2.0 mol/L in diethyl ether, 0.73 mL, 1.46 mmol) and the reactionmixture was stirred at room temperature for 1 h. The reaction mixturewas concentrated in vacuo and taken up in dichloromethane. MP-carbonate(464 mg, 1.458 mmol) was added and the reaction mixture was stirredovernight. The mixture was filtered and concentrated in vacuo andpurified by reverse-phase HPLC to yield the title compound (25.4 mg,44%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.64 (s, 1H), 9.33(dd, J=7.0, 1.6 Hz, 1H), 8.66-8.63 (m, 2H), 8.52 (s, 1H), 8.31 (s, 1H),8.24 (s, 2H), 8.11 (dd, J=8.7, 2.5 Hz, 1H), 8.03 (d, J=2.5 Hz, 1H),7.99-7.94 (m, 1H), 7.64 (d, J=8.7 Hz, 1H), 7.42 (t, J=72.5 Hz, 1H), 7.28(dd, J=7.0, 4.3 Hz, 1H), 4.02 (t, J=6.0 Hz, 2H), 3.95 (s, 3H), 2.97 (t,J=7.0 Hz, 4H), 1.87-1.76 (m, 2H). LC/MS (Method X, ESI): [M+H]+=598.2R_(T)=3.07 min.

Example 40

N-(3-(2-(difluoromethoxy)-5-(phenylsulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis ofN-(3-(2-(difluoromethoxy)-5-(phenylthio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 3, 200 mg, 0.43 mmol), thiophenol (57 mg, 0.053 mL, 0.52mmol), XantPhos Pd G2 (38.3 mg, 0.0432 mmol) and XantPhos (25 mg, 0.0432mmol) in toluene (11 mL) cooled to 0° C. was added NaH (34.5 mg, 0.86mmol) and the reaction mixture was stirred for 30 min at 0° C., 1 h atroom temperature, and then heated to 90° C. for 2 h. The reactionmixture was diluted with dichloromethane, filtered through Celite,eluting with dichloromethane, and the filtrate was concentrated invacuo. The crudeN-(3-(2-(difluoromethoxy)-5-(phenylthio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamidewas used without further purification. LC/MS (Method W, ESI):[M+H]+=593.1 R_(T)=1.34 min.

Step 2: Synthesis ofN-(3-(2-(difluoromethoxy)-5-(phenylsulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-phenylsulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(213 mg, 0.432 mmol) in dichloromethane (4.0 mL) was added3-chloroperbenzoic acid (203 mg, 0.91 mmol) and the reaction was stirredat room temperature for 2 d. 3-chloroperbenzoic acid (68 mg, 0.302 mmol)was added, and after 1 h a second portion was added (25 mg). After 1 h,the reaction mixture was diluted with dichloromethane and poured into aseparatory funnel containing saturated aqueous sodium bicarbonate. Thelayers were separated and the aqueous layer was extracted withdichloromethane (2×). The combined organic layers were dried over sodiumsulfate, filtered and concentrated in vacuo. The residue was purified byreverse-phase HPLC to yield the title compound (107.2 mg, 47%) as awhite solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.61 (s, 1H), 9.32 (dd, J=7.0,1.5 Hz, 1H), 8.64 (s, 1H), 8.62 (dd, J=4.3, 1.7 Hz, 1H), 8.31 (s, 1H),8.15 (dd, J=8.8, 2.5 Hz, 1H), 8.06 (d, J=2.5 Hz, 1H), 8.03-7.93 (m, 2H),7.71-7.22 (m, 6H), 3.95 (s, 3H). LC/MS (Method X, ESI): [M+H]+=525.1R_(T)=4.58 min.

Example 44

N-(3-(2-(difluoromethoxy)-5-((1-methyl-2-oxo-1,2-dihydropyridin-4-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis ofN-[3-[2-(difluoromethoxy)-5-[(1-methyl-2-oxo-4-pyridyl)sulfanyl]phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a vial was addedN-[3-[2-(difluoromethoxy)-5-sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 3, 50 mg, 0.12 mmol) and XantPhos Pd G3 (9.60 mg, 0.0096mmol). N,N-dimethylformamide (1.5 mL) was added followed by4-bromo-1-methylpyridin-2(1H)-one (47.5 mg, 0.240 mmol) andN,N-diisopropylethylamine (46.6 mg, 0.0628 mL, 0.360 mmol), and nitrogenwas bubbled through the reaction mixture for 3 min. The reaction mixturewas heated to 180° C. in a microwave reactor for 30 min. The reactionmixture was quenched by the addition of saturated aqueous ammoniumchloride and extracted with EtOAc (3×). The combined organic layers werewashed with brine, dried over sodium sulfate, filtered and concentratedin vacuo to giveN-[3-[2-(difluoromethoxy)-5-[(1-methyl-2-oxo-4-pyridyl)sulfanyl]phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide.

Step 2: Synthesis ofN-(3-(2-(difluoromethoxy)-5-((1-methyl-2-oxo-1,2-dihydropyridin-4-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-[(1-methyl-2-oxo-4-pyridyl)sulfanyl]phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(63 mg, 0.120 mmol) in dichloromethane (2.0 mL) was added3-chloroperbenzoid acid (108 mg, 0.48 mmol) and the reaction was stirredat room temperature for 15 min. The reaction mixture was diluted withdichloromethane and poured into a separatory funnel containing saturatedaqueous sodium bicarbonate. The layers were separated and the aqueouslayer was extracted with dichloromethane (2×). The combined organiclayers were dried over sodium sulfate, filtered and concentrated invacuo. The residue was further purified by RP-HPLC to yield the titlecompound (29.2 mg, 44%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ9.63 (s, 1H), 9.33 (dd, J=6.9, 1.6 Hz, 1H), 8.66-8.63 (m, 2H), 8.32 (s,1H), 8.22 (dd, J=8.8, 2.5 Hz, 1H), 8.09 (d, J=2.5 Hz, 1H), 7.90 (d,J=7.1 Hz, 1H), 7.68 (d, J=8.7 Hz, 1H), 7.48 (t, J=72.3 Hz, 1H), 7.28(dd, J=7.0, 4.3 Hz, 1H), 6.94 (d, J=2.2 Hz, 1H), 6.60 (dd, J=7.1, 2.1Hz, 1H), 3.96 (s, 3H), 3.40 (s, 3H). LC/MS (Method X, ESI): [M+H]+=556.1R_(T)=3.18 min.

Example 15

(S)—N-(3-(2-(difluoromethoxy)-5-((2-hydroxypropyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis of(S)—N-(3-(2-(difluoromethoxy)-5-((2-hydroxypropyl)thio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(Intermediate 8, 950 mg, 2.28 mmol) in methanol (10 mL) was added(S)-(−)-propylene oxide (250 mg, 4.3 mmol) and DIEA (900 mg, 6.98 mmol)and the mixture was stirred at 80° C. for 3 h. After cooling to roomtemperature, the reaction mixture was concentrated under vacuum. Theresidue was purified by reverse phase HPLC [mobile phase A: water (0.1%NH₄HCO₃), mobile phase B: acetonitrile; gradient: 10% B to 40% B in 50min] to giveN-[3-[2-(difluoromethoxy)-5-[(2S)-2-hydroxypropyl]sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(730 mg, 1.54 mmol, 67% yield) as a light yellow solid. LC/MS (Method 0,ESI): [M+H]+=475.2 R_(T)=1.43 min.

Step 2: Synthesis of(S)—N-(3-(2-(difluoromethoxy)-5-((2-hydroxypropyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-[(2S)-2-hydroxypropyl]sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(730 mg, 1.54 mmol) in methanol (10 mL) was added a solution of oxone(2.0 g, 3.25 mmol) in water (5.0 mL) and the mixture was stirred at roomtemperature for 3 h. The organic solvent was removed under vacuum and 10mL of water was added. The solid was collected by filtration. The filtercake was washed with water (5.0 mL×5) and dried under vacuum. Theresidue was purified by preparatory HPLC (column: XBridge Prep OBD C18Column, 30×150 mm 5 um; mobile phase A:water (10 mmol/L NH₄HCO₃), mobilephase B: ACN; flow rate:60 mL/min; gradient:16% B to 37% B in 8 min; 254nm; R_(T) 1:7.45 min). The product was stirred in 3.0 mL of ethanolovernight at room temperature to giveN-[3-[2-(difluoromethoxy)-5-[(2S)-2-hydroxypropyl]sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(783 mg, 1.52 mmol) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ9.69 (s, 1H), 9.35 (dd, J=6.8, 1.6 Hz, 1H), 8.70-8.68 (m, 2H), 8.34 (s,1H), 8.09-8.04 (m, 2H), 7.68-7.29 (m, 3H), 4.90 (d, J=5.6 Hz, 1H),4.10-4.04 (m, 1H), 3.97 (s, 3H), 3.50-3.40 (m, 2H), 1.12 (d, J=6.0 Hz,3H). LC/MS (Method 0, ESI): [M+H]+=507.2 R_(T)=1.26 min.

Example 25

(R)—N-(3-(2-(difluoromethoxy)-5-((2-hydroxypropyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis of(R)—N-(3-(2-(difluoromethoxy)-5-((2-hydroxypropyl)thio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(1.10 g, 2.64 mmol) in methanol (10 mL) was added (R)-(+)-propyleneoxide (300 mg, 5.17 mmol) and DIEA (1.00 g, 7.75 mmol) and the reactionmixture was stirred at 80° C. for 3 h. After cooling to roomtemperature, the solvent was removed under vacuum. The residue waspurified by reverse phase HPLC [mobile phase A: water (0.1% NH₄HCO₃),mobile phase B: acetonitrile; gradient: 10% B to 40% B in 50 min] togiveN-[3-[2-(difluoromethoxy)-5-[(2R)-2-hydroxypropyl]sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(780 mg, 1.64 mmol, 62% yield) as an off-white solid. LC/MS (Method 0,ESI): [M+H]+=475.2 R_(T)=1.43 min.

Step 2: Synthesis of(R)—N-(3-(2-(difluoromethoxy)-5-((2-hydroxypropyl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[2-(difluoromethoxy)-5-[(2R)-2-hydroxypropyl]sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(780 mg, 1.64 mmol) in methanol (10 mL) was added a solution of oxone(2.0 g, 3.26 mmol) in water (5.0 mL), and the mixture was stirred atroom temperature for 3 h. The organic solvent was removed under vacuum.Water (10 mL) was added and the solid was collected by filtration. Thefilter cake was washed with water (5.0 mL×5) and then dried undervacuum. The residue was purified by prep. HPLC (column: XBridge Prep OBDC18 Column, 30×150 mm 5 um; mobile phase A:water (10 mmol/L NH₄HCO₃),mobile phase B:ACN; flow rate:60 mL/min; gradient:16% B to 37% B in 8min; 254 nm; R_(T) 1:7.45 min). The product was stirred in 3.0 mL ofethanol overnight at room temperature to giveN-[3-[2-(difluoromethoxy)-5-[(2S)-2-hydroxypropyl]sulfonyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(771 mg, 1.51 mmol, 92% yield) as an off-white solid. LC/MS (Method 0,ESI): [M+H]+=507.2 R_(T)=1.26 min. ¹H NMR (400 MHz, DMSO-d₆) δ 9.69 (s,1H), 9.35 (dd, J=6.8, 1.6 Hz, 1H), 8.70-8.68 (m, 2H), 8.34 (s, 1H),8.10-8.04 (m, 2H), 7.68-7.29 (m, 3H), 4.90 (d, J=5.6 Hz, 1H), 4.10-4.04(m, 1H), 3.97 (s, 3H), 3.50-3.40 (m, 2H), 1.12 (d, J=6.0 Hz, 3H).

Examples 23 & 24

(R)—N-(3-(2-(difluoromethoxy)-5-((1-hydroxypropan-2-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide&(S)—N-(3-(2-(difluoromethoxy)-5-((1-hydroxypropan-2-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamideStep 1: Synthesis ofN-(3-(2-(difluoromethoxy)-5-((1-hydroxypropan-2-yl)thio)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

To a solution ofN-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(2.00 g, 4.32 mmol) in 1,4-dioxane (100 mL) was added Pd₂(dba)₃ (396 mg,0.431 mmol), Xantphos (500 mg, 0.864 mmol) and DIPEA (1.67 g, 13.0 mmol)at room temperature under nitrogen. The resulting solution was stirredfor 15 minutes at RT and heated to 90° C. 2-Sulfanylpropan-1-ol (580 mg,6.29 mmol) was added to the reaction mixture and the resulting mixturewas stirred at 90° C. for 10 h. The reaction was cooled to roomtemperature and the suspension was filtrated through Celite. Thefiltrate was concentrated under vacuum. The residue was purified byflash chromatography on silica gel eluting with DCM/EA (0%-100%) toaffordN-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-ethyl)sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(1.74 g, 3.67 mmol, 85% yield) as an off-white solid. LC/MS (Method I,ESI): [M+H]+=507.2 R_(T)=0.97 min.

Step 2: Synthesis ofN-(3-(2-(difluoromethoxy)-5-((1-hydroxypropan-2-yl)sulfonyl)phenyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

A solution ofN-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-ethyl)sulfanyl-phenyl]-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide(850 mg, 1.79 mmol) in methanol (8.0 mL) was stirred at 0° C. for 5 min.Then a solution of oxone (2.20 g, 3.58 mmol) in water (8.0 mL) was addedat 0° C. The reaction solution was stirred at room temperature for 1 h.The reaction mixture was concentrated and filtered. The filter cake wasmixed with DCM (10 mL) and filtered. The filtrate was concentrated undervacuum to afford the product (779 mg, 1.54 mmol, 86% yield). Thisreaction was repeated on the same scale and combined to afford 1.10 g(61% yield of racemate). The combined product was purified by chiral-SFCwith the following conditions: column: Lux Sum Cellulose-4, 5*25 cm, 5μm; mobile phase A: CO₂, mobile phase B: EtOH:ACN=2:1 (2 mM NH₃—MeOH);flow rate:150 mL/min; gradient: 40% B; 220 nm; R_(T)1: 9.03 min; R_(T)2:10.79 min. Fractions containing the two enantiomers were combined andconcentrated, and both were recrystallized by stirring in 2 mLisopropanol over 2 days at room temperature. The products were isolatedby filtration to afford the title compounds, with stereochemicalassignments made arbitrarily.

Peak 1:(R)-(N-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-ethyl)sulfonyl-phenyl)-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide,341 mg) was obtained as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ9.67 (s, 1H), 9.34 (dd, J=7.0, 1.6 Hz, 1H), 8.79-8.55 (m, 1H), 8.66 (s,1H), 8.32 (s, 1H), 8.17-7.85 (m, 2H), 7.79-7.16 (m, 3H), 4.96 (t, J=5.6Hz, 1H), 3.96 (s, 3H), 3.70 (m, 1H), 3.60-3.27 (m, 2H), 1.22 (d, J=6.7Hz, 3H). LC/MS (Method X, ESI): [M+H]+=507.1 R_(T)=3.59 min.

Peak2:(S)-(N-[3-[2-(difluoromethoxy)-5-(2-hydroxy-1-methyl-ethyl)sulfonyl-phenyl)-1-methyl-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide,411 mg) was obtained as an off-white. ¹H NMR (400 MHz, DMSO-d₆) δ 9.67(s, 1H), 9.34 (dd, J=7.0, 1.6 Hz, 1H), 8.79-8.45 (m, 2H), 8.32 (s, 1H),8.17-7.86 (m, 2H), 7.86-7.16 (m, 3H), 4.96 (t, J=5.5 Hz, 1H), 3.96 (s,3H), 3.70 (m, 1H), 3.60-3.28 (m, 2H), 1.22 (d, J=6.7 Hz, 3H). LC/MS(Method X, ESI): [M+H]+=507.1 R_(T)=3.56 min.

Assays Test Agents

Test agent samples were provided as solutions at a concentration of 10mM in dimethyl sulfoxide (DMSO) and were stored in the dark at roomtemperature before use.

JAK1 and JAK2 Biochemical Assays

The in vitro biochemical assays quantify JAK-catalyzed phosphorylationof a synthetic peptide, as detected using a LabChip® EZ Reader IImicrofluidic mobility shift instrument (PerkinElmer; Waltham, Mass.).The substrate peptide Y-1B has the sequence 5-FAM-VALVDGYFRLTT-NH₂. Y-1Bis fluorescently labeled on the N-terminus with 5-FAM(5-carboxyfluorescein) and contains a single tyrosine residue (Y) thatcan be phosphorylated by JAK activity. The substrate peptide stock isprepared in DMSO at 5 mM. Purified recombinant human JAK1 kinase domainprotein (Residues 854-1154) was expressed in insect cells and procuredfrom Proteros Biostructures GmbH (Martinsried, Germany). Recombinanthuman JAK2 kinase domain protein (Residues 812-1132) was expressed ininsect cells and purified at Genentech, Inc. (South San Francisco,Calif.).

The kinase reaction mixtures contained 100 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH7.2), 10 mM magnesium chloride, 0.015% Brij® 35, 4 mM dithiothreitol,1.5 μM Y-1B peptide substrate, 25 μM adenosine triphosphate (ATP), 1 nMtotal JAK1 or 0.2 nM total JAK2, and up to 1000 nM test compound in afinal concentration of 2% (volume to volume [v/v]) DMSO. In eachtitration experiment, test compound was tested in duplicate at each ofthe twelve concentrations. Blank reactions contained ATP, peptide, andDMSO, but no JAK or test compound, whereas uninhibited control reactionscontained ATP, peptide, JAK, and DMSO, but no test compound.

Peptide plus ATP mixture (24 μL) was added to 1 μL of test compound inDMSO (or DMSO alone). The reactions were initiated by adding 25 μL ofJAK enzyme to the inhibitor/peptide/ATP mixture before thoroughly mixingthe resultant solution. Reactions were incubated at room temperature(22° C.-23° C.) in a final volume of 50 μL per well in 384-well plates.After a 30-minute incubation, the reactions were stopped by adding 25 μLof 150 mM ethylenediaminetetraacetic acid in 100 mM HEPES buffer (pH7.2) containing 0.015% Brij 35 to each well.

In each reaction, the residual Y-1B substrate and the phospho-peptideproduct generated were separated using the EZ Reader II instrument.Electrophoretic separation of molecules of product from molecules ofsubstrate was achieved using downstream and upstream voltages of −500and −2600 V, respectively, at an operating pressure of −1.3 psi. The5-FAM group present on both the substrate and product peptides wasexcited at 488 nm, the fluorescence was detected at 530 nm, and the peakheights were reported.

Data Analysis

The extent (or percent) of conversion of substrate to product wascalculated from the corresponding peak heights in the electropherogramusing HTS Well Analyzer software, Version 5.2 (PerkinElmer), and thefollowing equation (Equation 1):

% conversion=[P÷(S+P)]×100  Equation 1

where S and P represent the peak heights of the substrate and product,respectively. After any baseline signal from blank wells containing noJAK was subtracted from the signal of all test wells, the % conversiondata were converted to fractional activity as shown in Equation 2, whereν_(i) and ν_(o) are the % conversion in the presence and absence of testcompound, respectively. The % conversion observed in the uninhibitedcontrol reactions containing JAK and DMSO vehicle, but no test compound,was defined to have fractional activity=1 (with no inhibitor present,ν_(i)=ν_(o)), whereas blank wells with no JAK were defined as havingfractional activity=0. Fractional activity was plotted against testcompound concentration and the data were fitted using XLfit software(IDBS; Guildford, United Kingdom) to a tight-binding apparent inhibitionconstant (K_(i) ^(app)) quadratic equation (see Equation 2) (Williams JW, Morrison J F. The kinetics of reversible tight-binding inhibition.Methods Enzymol 1979; 63:437-67.), which was used to calculatefractional activity and K_(i) ^(app)

$\begin{matrix}{{{Fractional}{activity}} = {\frac{v_{i}}{v_{o}} = {1 - \frac{\left( {\lbrack E\rbrack_{T} + \lbrack I\rbrack_{T} + K_{i}^{app}} \right) - \sqrt{\left( {\lbrack E\rbrack_{T} + \lbrack I\rbrack_{T} + K_{i}^{app}} \right)^{2} - {{4\lbrack E\rbrack}_{T}\lbrack I\rbrack}_{T}}}{{2\lbrack E\rbrack}_{T}}}}} & {{Equation}2}\end{matrix}$

where [E]_(T) and [I]_(T) are the total concentrations of active enzyme(initial estimates of 0.15 nM for JAK1 and 0.048 nM for JAK2) andinhibitor (the varied parameter), respectively. Finally, the K_(i) wascalculated from the K_(i) ^(app) by applying the competitive inhibitionrelationship (Equation 3)

K_(i)=K_(i) ^(app)/(1+[ATP]/K_(m) ^(app))  Equation 3

where [ATP] is the concentration of ATP=25 μM, K_(m) ^(app) is theapparent ATP Michaelis constant=32.1 μM for JAK1, and K_(m) ^(app)=11.7μM for JAK2. By applying the tight-binding Equation 2 to account for anydepletion of inhibitor, and the competitive-inhibition relationshipEquation 3, the sensitivity of the assay can extend at least to acalculated K_(i) of 0.008 nM for JAK1 and 0.0015 nM for JAK2.

Kinase Selectivity

The in vitro kinase selectivity of test agents was assessed at aconcentration of 1 μM in a panel of recombinant human kinase activityand binding assays, including cytoplasmic and receptor tyrosine kinases,serine/threonine kinases, and lipid kinases (SelectScreen® KinaseProfiling Services, ThermoFisher Scientific, Madison, Wis.). The kinaseactivity assays measure peptide phosphorylation (Z′-LYTE®) or ADPproduction (Adapta®) while the binding assays monitor displacement ofATP site binding probes)(LanthaScreen®). The ATP concentrations used inthe activity assays were typically within 2-fold of the experimentallydetermined apparent Michaelis constant (K_(m) ^(app)) value for eachkinase while the competitive binding tracer concentrations used in thebinding assays were generally within 3-fold of the experimentallydetermined dissociation constant (K_(d)) values. Inhibitors were testedin duplicate against each kinase and the mean % Inhibition values arereported. For kinases that were inhibited by close to or greater than50% at the initial 1-μM test concentration, 10-point inhibitortitrations using the same assays were carried out in order to determinethe inhibitor concentrations that caused 50% inhibition (IC₅₀). Thetotal JAK1 concentration used in this assay panel was 75 nM. If 100% ofthe 75 nM JAK1 protein were catalytically active, the limit of JAK1inhibitor sensitivity from the vendor's JAK1 assay would theoreticallybe an IC₅₀ value of 37.5 nM (one-half of the total enzymeconcentration). However, the SelectScreen® JAK1 assay generated JAK1IC₅₀ values for several inhibitors that are much lower than 37.5 nM andwhich are in agreement with our internal determinations. Thus, theactive JAK1 enzyme concentration in the SelectScreen® assay must be muchlower than the total nominal JAK1 protein concentration of 75 nM used inthe assay, and the observed sensitivity of this assay is much betterthan the theoretical sensitivity IC₅₀ limit of 37.5 nM.

Data Analysis

For fitting the data in concentration—kinase inhibition plots, theSelectScreen® Kinase Profiling Services used XLfit software (IDBS),Model No. 205 (sigmoidal concentration—response model), which is afour-parameter logistic fit model described by Equation 4

y=A+{(B−A)÷[1+(C÷x)^(D)]}  Equation 4

where x is the inhibitor concentration, y is the observed % inhibition,A is the minimum y-value, B is the maximum y-value, C is the IC₅₀ value,and D is the Hill slope. In certain cases, a three-parameter logisticfit was used. For example, if the plateau of the curve at infinitely lowinhibitor concentration did not fit between −20% and 20% inhibition,that lower plateau was set to 0% inhibition, whereas if the plateau ofthe curve at infinite inhibitor concentration did not fit between 70%and 130% inhibition, that upper plateau was set to 100% inhibition.

TF-1 Cell Line Phospho-STAT JAK1 and JAK2 Pathway Selectivity Assays

TF-1 human erythroleukemia cells (ATCC®; Manassas, Va.; Catalog No.CRL-2003™) were grown in Roswell Park Memorial Institute (RPMI) mediumsupplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 ng/mLgranulocyte-macrophage colony-stimulating factor, 1×non-essential aminoacids (NEAA), and 1 mM sodium pyruvate. The day before the assay, thecultures were transferred to Opti-MEM™, 1×NEAA, 1 mM sodium pyruvate,and 0.5% charcoal-stripped FBS (starve medium). Inhibitor stocksolutions (5 mM in DMSO) were serially diluted 1:2 in DMSO to generate a10-point concentration titration (at 500×test concentration), which wasfurther diluted by a 50-fold dilution in Assay Medium (RPMI containing1×NEAA and 1 mM sodium pyruvate) to generate a 10×concentrationtitration (in 2% DMSO). The cells (300,000 cells/well in 35 μL of AssayMedium) were seeded in 384-well Greiner plates. Diluted inhibitor at10×concentration (5 μL) was added to the cells and the plates wereincubated for 30 minutes at 37° C. in a humidified incubator. Cells werestimulated with the human recombinant cytokine at the respective EC %concentrations, as previously determined for each individual lot. Forthe phosphorylated signal transducer and activator of transcription 6(P-STATE) TF-1+Interleukin-13 (IL-13) assay, 10 μL of 250 ng/mL IL-13(R&D Systems; Minneapolis, Minn.) was added to the cells, which werethen incubated for 10 minutes at 37° C. For the P-STAT5TF-1+Erythropoietin (EPO) assay, 10 μL of 110 IU/mL EPO (Gibco LifeTechnologies, Catalog No. PHC2054) was added to the cells, which werethen incubated for 30 min at 37° C. For both assays, the incubation wasfollowed by addition to the cells of 5 μL of ice-cold 10×cell lysisbuffer (Cell Signaling Technologies; Danvers, Mass.; Catalog No. 9803S)containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Assay plates werefrozen at −80° C. for a minimum of 1 hour. In the IL-13 assay, P-STAT6was measured by coating goat anti-rabbit (GAR) plates (Meso ScaleDiscovery [MSD]; Rockville, Md.; Catalog No. MSD L21RA-1) with rabbitanti-human total STAT6 antibody (Cell Signaling Technologies; CatalogNo. 9362S), incubating the cell lysates in the coated plates overnightat 4° C., and then detecting with mouse anti-P-STAT6 (Tyr641) Clone16E12 antibody (MilliporeSigma; Burlington, Mass.; Catalog No. 05-590,custom labeled by MSD with SULFO-tag) using standard MSD plateprocessing, washing, and detection protocols. In the EPO assay, P-STAT5was detected using the phospho-STAT5a,b Whole Cell Lysate Kit (MSD;Catalog No. K150IGD-1). The electrochemiluminescence (ECL) signal ofwells was read on the MESO SECTOR S600 (MSD) reader.

Data Analysis

Data analysis was performed by subtracting the negative control(cytokine stimulated and 20 control inhibitor-treated cells) mean ECLvalue from the ECL value of all wells, determining percent of controlfor test compound well ECL values relative to the positive control(cytokine stimulated and DMSO-treated cells) mean ECL value, anddetermining the IC₅₀ for test compounds with a four-parameter logisticfit model as shown in Equation 4.

P-STAT6 BEAS-2B+IL-13 Cell Assay

In order to study the effect of JAK1 inhibitors in a cell line that isrelevant to the cell biology of human asthma, an IL-13—stimulated STAT6phosphorylation assay in the human lung bronchial epithelial BEAS-2Bcell line was developed.

BEAS-2B cells (ATCC® CRL-9609™) were grown in Bronchial EpithelialGrowth Medium (BEGM) (Lonza Catalog No. CC-3170; Walkersville, Md.; orPromoCell Catalog No. C-21060; Heidelberg, Germany). Test compound stocksolutions (0.5 mM in DMSO) were serially diluted 1:2 in DMSO to generatea 10-point concentration curve (at 500×test concentration), which wasfurther diluted by a 50-fold dilution step in BEGM to generate a10×concentration curve (in 2% DMSO). Cells were plated at 100,000cells/well in 200 μL of BEGM in 96-well plates and incubated for 48hours at 37° C. in a humidified incubator. Medium was aspirated from thecells and replaced with 70 μL of fresh BEGM. Diluted test compound (10μL; or 2% DMSO in assay medium) was added to the cells, and the plateswere incubated for 1 hour at 37° C. in a humidified incubator. Twenty μLof 250 ng/mL human recombinant IL-13 (Bio Techne Catalog No. 213-ILB)was then added to the cells and incubated for 15 minutes at 37° C.Medium was aspirated from the cells and 60 μL of ice-cold 1×cell lysisbuffer (Cell Signaling Technologies; Catalog No. 9803S) containing 1 mMPMSF was added to the cells. Assay plates were incubated at −80° C. forat least 1 hour. P-STAT6 was measured by coating GAR plates (MSD;Catalog No. L45RA-1) with rabbit anti-human total STAT6 antibody (CellSignaling Technologies; Catalog No. 9362S), incubating the cell lysatesin the coated plates overnight at 4° C., and then detecting with mouseanti-phospho-STAT6 (Tyr641) Clone 16E12 antibody (Millipore; Catalog No.05-590, custom labeled by MSD with SULFO-tag) using standard MSD plateprocessing, washing, and detection protocols. Plates were read on theMESO SECTOR S600.

Data Analysis

Data analysis was performed by subtracting negative control values fromall wells and determining percent of control using the positive controlvalues; the IC₅₀ was determined with a four-parameter logistic fit modelas shown in Equation 4.

Cell Cytotoxicity Assays

A549 (ATCC® CCL-185™), Jurkat clone E6-1 (ATCC® TIB-152™) and HEK-293T(ATCC® CRL-1573™) cells maintained at a sub-confluent density in T175flasks were used. Cells in exponential growth phase were plated (450cells in 45 μL of medium) in Greiner 384-well black/clear tissue culturetreated plates (Greiner Catalog No. 781091). After dispensing cells,plates were allowed to equilibrate at room temperature for 30 minutes,after which time the cell plates were placed overnight in a 37° C. CO₂and humidity-controlled incubator. The following day, cells were treatedwith test agent diluted in 100% DMSO (0.5% final DMSO concentration oncells) with a 10-point titration and a top concentration of 50 μM. Cellsand compounds were then incubated for 72 hours in a 37° C. CO₂ andhumidity-controlled incubator, after which time cell viability wasmeasured by adding CellTiter-Glo® (Promega G7572) reagent to all wells.Plates were incubated at room temperature for 20 minutes and then thewell luminescence was read on an EnVision plate reader (Perkin ElmerLife Sciences).

Data from the above JAK1 and JAK2 assays for the compounds of Table 1are shown in Table 2 below.

TABLE 2 Cellular Cellular Cellular JAK1 JAK1 JAK2 Enzyme Enzyme PSTAT6PSTAT6 PSTAT5 JAK1 JAK2 BEAS2B+ TF-1+ TF-1+ et. Ki Ki IL13 IL13 EPOLC/MS ime

(nM) (nM) IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) method (min) /z 0.56 0.25 31 6.312 D .44

91.2 0.5 0.2 15 14 12 A .53

03.2 2 0.66 58 A .15

18.3 1.4 0.6 180 A .13

04.2 0.43 0.19 35 16 29 A .27

93.2 0.48 0.22 18 A .42

77.2 0.88 0.49 32 21 22 A .13

20.3 0.82 0.48 92 A .12

92.2 0.27 0.18 15 A .56

99.2 0 0.95 0.6 52 A .15

18.3 1 0.21 0.12 17 A .52

41.3 2 0.24 0.14 18 8.7 9.1 A .65

55.3 3 0.4 0.19 33 34 28 D .1

05.2 4 0.49 0.22 23 A .4

07.2 5 0.43 0.16 20 T .94

07.2 6 0.32 0.19 15 A .47

26.3 7 0.22 0.1 14 A .36

63.1 8 0.43 0.4 290 B .14

68.2 9 4 1.7 120 A .15

32.3 0 1.5 0.65 65 A .16

32.3 1 0.22 0.16 14 A .49

04.3 2 0.46 0.19 23 4.6 14 A .47

89.2 3 0.26 0.13 14 20 22 A .33

07.2 4 0.46 0.19 30 A .33

07.2 5 0.48 0.2 20 20 39 A .33

07.2 6 0.16 0.097 11 A .39

78.2 7 0.59 0.26 99 A .13

46.2 8 0.24 0.14 9.5 3.1 26 A .43

88.2 9 0.58 0.37 25 A .61

24.1 0 0.22 0.14 11 D .47

92.1 1 0.84 0.38 51 B .2

64.1 2 1.1 0.62 120 B .41

33.2 3 0.4 0.23 99 B .41

33.2 4 0.31 0.29 21 A .68

35.2 5 0.3 0.19 24 5.8 14 X .96

29.2 6 1.8 1.1 71 120 U .22

32.1 7 0.69 0.54 46 51 38 U .22

32.1 8 0.34 0.25 89 X .98

73.2 9 1.5 0.53 230 X .33

33.1 0 0.21 0.11 24 3.1 6.4 X .67

25.1 1 0.18 0.1 37 15 34 X .71

15.1 2 0.42 0.24 30 2.6 21 X .15

26.1 3 0.5 0.27 1000 X .75

42.1 4 0.52 0.34 29 14 15 X .18

56.1 5 0.71 0.35 35 10 38 D .49

14.1 6 0.26 0.087 49 27 61 B .19

90.1 7 0.31 0.33 210 X .98

58.2 8 0.52 0.39 130 X .11

72.2 9 0.5 0.37 28 20 34 X .02

86.2 0 1.7 1.9 50 27 160 X .97

46.2 1 0.39 0.21 100 X .82

30.1 2 2.2 1 96 X

44.2 3 0.94 0.19 50 X .07

98.2 4 1.2 0.21 140 X .1

41.2 5 0.93 0.98 71 X .19

24.2 6 0.71 0.76 56 X .16

26.2 7 0.57 0.54 54 X .15

26.2 8 0.89 0.86 40 24 32 X .12

00.2 9 0.53 0.54 32 13 28 X .09

00.2 0 1.1 2.5 44 24 170 X .06

72.2 1 0.81 0.77 34 190 200 X .06

84.2 2 2 0.93 150 X .83

92.1 3 0.58 0.28 71 X .53

07.1 4 1.9 0.84 210 X .8

92.2 5 0.72 0.24 72 X .66

29.1 6 0.55 0.18 380 X .5

15.1 7 0.48 0.24 25 X .97

27.1 8 0.6 0.25 62 X .78

21.2 9 77 X .72

21.2 0 1 0.33 75 X .79

21.2 1 1.4 0.46 110 X .78

21.2 2 0.94 1.5 31 X .89

46.2 3 0.63 0.21 53 A .32

22.2

indicates data missing or illegible when filed

As can be seen from Table 2, the compounds of the invention are allhighly active in the biochemical JAK1 and JAK2 assays as well as, inmost instances, one or more of the cell-based assays for JAK1 and JAK2.Several of the compounds are substantially equipotent for JAK1 and JAK2.

Table 3 below provides mouse lung tissue binding (LTB) data (% bound)and cytotoxity data for selected compounds of Table 1.

TABLE 3 Table 3 Mouse Cytotox K- Cytotox Cytotox LTB % 293 EC₅₀ (μM)Jurkat EC₅₀ (μM) A549 EC₅₀ (μM) 88.1 27.5 38.5 25.5 80.1 10.2 23 45.56.05 21.5 6.75 78.8 6.9 2.1 4.1 2.2 3.5 1.66 1 3.7 5.5 6.4 2 97.1 36 5050 3 85 31 50 44 4 80.1 32 50 50 5 85.7 25 35 25 6 87.3 7.6 6.2 7.4 785.8 7.3 11 8.7 1 83 4.8 11 7.5 2 88.7 43 50 50 3 82.1 11 24 28 4 81.722 37 50 5 85 48 50 50 6 86.9 5.8 12 11 8 70.8 24 39 50 0 83.1 2.4 9.26.1 5 86.2 17 21 22 7 90.1 31 50 50 0 95.6 50 50 50 1 91.2 28 35 50 486.5 7 11 24 5 87.9 50 50 50 6 89.9 50 50 50 8 91.3 9 84.4 14 24 33 8 1932 50 6 91.4 10 24 50 9 14 30 43 3 80.5 20 35 50 9 78.4 2 20 49 50 381.8 2.9 8.4 50

From the data in Table 3, it was discovered that 1-methyl-pyrazolecompounds (i.e., R¹ in Formulas (I)-(III) is methyl) have surprisinglylower cytotoxicity across K-293, Jurkat and A549 cell line assays whencompared to the corresponding desmethyl (R¹ is hydrogen) analogs. Table4 below provides comparisons of methyl-desmethyl analog pairs (R¹=methylvs R¹=hydrogen) for Examples 6, 9 and 17 of Tables 1 and 2.

TABLE 4 Cytotox Cytotox Cytotox K- Jurkat A549 293 EC50 EC50 EC50 (μM)(μM) (μM) Example 6 in Table 1.

6.05 21.5 6.75 Example. 192 in WO2017089390

0.436667 0.79 2.366667 Example 9 in Table 1.

2.2 3.5 1.66

0.038 0.16 0.26 Example 17 in Table 1

7.3 11 8.7 Example 4 in WO2017089390

2.1 6.2 22

From the data in Table 3, it was also discovered that the sulfonecompounds of the invention exhibit surprisingly improved lung tissuebinding when compared to corresponding sulfide analogs. Table 5 belowprovides comparisons of sulfone-sulfide analog pairs (R¹=methyl vsR¹=hydrogen) for Examples 13, 28 and 35 of Tables 1 and 2

TABLE 5 Lung tissue binding, % Example 13 in Table 1

85.8

97.4 Example 28 in Table 1

70.8

96.9 Example 35 in Table 1

86.2

97

The sulfone compounds of the invention also exhibit lower LogD, andincreased kinetic solubility compared to the corresponding sulfideanalogs.

From the data in Tables 2 and 3, it was also discovered that compoundsof the invention wherein R¹ is methyl and R² is hydroxyalkyl oralkoxyalkyl exhibit good, balanced affinity for both JAK1 and JAK2, andalso have relatively low lung tissue binding and cytotoxicity.

Animal Models

Mouse House Dust Mite (HDM) Model

Seven to eight week old female C57BL/6J mice purchased from JacksonWest. Mice are immunized on day 0 & 14 with intraperitonealadministration of House Dust Mite (HDM, D. Pteronyssinus, purchased fromGreer Laboratories, normalized to 0.918 ug DerP1 content per mouse)mixed with 2 mg of alum (Thermo Scientific) diluted in sterile PBS. Ondays 21 & 24, mice were challenged with HDM (again normalized for 0.918ug DerP1 content) in PBS, dosed by intra-tracheal inhalation. Prior toeach inhaled HDM challenge (and in a subset of groups, also on days 22 &23), animals receive test compound via nose-only inhalation (using drypowder inhalation equipment from Electro-Medical Measurement Systems(EMMS), including a Wright dust feeder and a 4-layer/24-port or2-layer/12-port, directed flow, nose-only inhalation tower) ending 1hour prior to challenge. Control animals receive air-only nose onlyinhalation. 24 hours after the final treatment, mice are bledretro-orbitally for plasma PK, and then euthanized by CO₂ inhalation.Post-euthanasia, BAL fluid is collected for total (by FACS, using aknown quantity of spike-in reference beads) and differential (by WrightGiemsa—stained cytospin) cell counts. Lungs and spleens are collected,weighed, and frozen for PK. There were 5 or 6 animals per group.

In addition, to validate lung-delivered dose, PK satellite groups of 3naïve animals each are dosed with test compound via nose-only inhalationfor a single day or for four consecutive days. Directly after the finalinhalation dosing, PK satellite animals are bled retro-orbitally forplasma PK, and then euthanized by CO₂ inhalation. Lungs and spleens arecollected and weighed for PK analysis.

Rat OVA Model

Six week old male Brown Norway rats from Charles River-Kingston. Ratsare immunized on day 0 with intraperitoneal administration of 150 ug OVA(Sigma) mixed with 40 mg of alum (Thermo Scientific) diluted in sterilePBS. 28 days after sensitization, rats are challenged with 2% OVA in PBSaerosolized via a nebulizer for 30 minutes for three consecutive days.Prior to each OVA challenge, animals receive JAK1/JAK2 test compound vianose-only inhalation (using dry powder inhalation equipment fromElectro-Medical Measurement Systems (EMMS), including a Wright dustfeeder and a 4-layer, 24-port, directed flow, nose-only inhalationtower) ending 1 hour prior to challenge. Control animals receive eitherMCT buffer orally, or air-only nose only inhalation. 24 hours after thefinal treatment, rats are euthanized by CO₂ inhalation. They are bledfrom the abdominal aorta for plasma PK and whole blood FACS analysis.Post-euthanasia, BAL fluid is collected for total (by FACS, using aknown quantity of spike-in reference beads) and differential (by WrightGiemsa—stained cytospin) cell counts. Lungs are collected, weighed, andfrozen for PK. Spleens are weighed and cut in half for PK and for FACSanalysis. Blood and spleen samples are analyzed by FACS for total cellcounts and % NK cells (CD161a positive). There are 6 animals per group,except for the naïve control group, which contains 5 animals.

In addition, to validate lung-delivered dose, PK satellite groups of 3naïve animals each received JAK1/JAK2 test compound via nose-onlyinhalation for a single day or for three days. Directly after the finalinhalation dosing, PK satellite animals are euthanized by CO₂inhalation. They are bled from the abdominal aorta for plasma PK. Lungsand spleens was collected and weighed for PK analysis.

Plasma and lung levels of test compounds and ratios thereof aredetermined in the following manner. BALB/c mice from Charles RiverLaboratories are used in the assay. Test compounds are individuallyformulated in 0.2% Tween 80 in saline and the dosing solution isintroduced into the trachea of a mouse by oral aspiration. At varioustime points (typically 0.167, 2, 6, 24 hr) post dosing, blood samplesare removed via cardiac puncture and intact lungs are excised from themice. Blood samples are centrifuged (Eppendorf centrifuge, 5804R) for 4minutes at approximately 12,000 rpm at 4° C. to collect plasma. Lungsare padded dry, weighed, and homogenized at a dilution of 1:3 in sterilewater. Plasma and lung levels of test compound are determined by LC-MSanalysis against analytical standards constructed into a standard curvein the test matrix. A lung to plasma ratio is determined as the ratio ofthe lung AUC in micro g hr/g to the plasma AUC in micro g hr/mL, whereAUC is conventionally defined as the area under the curve of testcompound concentration vs. time.

Pharmacokinetics in Plasma and Lung in Mouse

The pharmacokinetics of a compound is determined in female Balb/c micefollowing administration of a target dose of 0.3 mg/kg formulated in0.2% Tween 80 in saline by single intra-nasal (IN) bolussolution/suspension administration. 7-8 Week old female Balb/c mice amaybe purchased from Charles River. Mice are housed under specificpathogen-free conditions until used in a study.

Animals are not fasted before dosing. Blood samples are taken from 3animals per time-point at 0.083, 2, 7 and 24 hours post-dose, underanesthesia (intraperitoneal injection of pentobarbitone), via cardiacpuncture into EDTA-coated microtainers. Blood samples are centrifuged(1500 g, 10 min at 4° C.) to separate plasma. Plasma samples frozen atapproximately −80° C. After intra-nasal dosing, prior to lung perfusion,the spleens are removed, weighed and snap frozen. Following confirmationof death, the lungs of the dosed animals are perfused with chilled PBSto remove residual blood from the pulmonary vasculature. The lungs arethen excised and weighed (all weights recorded). All tissue samples arefrozen by immersion in liquid nitrogen. Tissue samples are stored frozen(ca. −80° C.) until analysis.

Prior to PK analysis defrosted tissue samples (spleen and lung) areweighed and homogenised following the addition of 4 mL HPLC grade waterfor each gram of tissue, using an Omni-Prep Bead Ruptor (Omni Inc.,Kennesaw, Ga.) at 4° C. Plasma and tissue homogenate samples areextracted using protein precipitation with four volumes of acetonitrilecontaining Tolbutamide (200 ng/mL) or Labetalol (100 ng/mL) as internalstandard. Samples are mixed and centrifuged at 3200 g and 4° C. for 30minutes to remove precipitated proteins, and the supernatant dilutedappropriately (e.g. 1:1, v/v) with HPLC grade water in a 96-well plate.Representative aliquots of plasma, spleen and lung samples are assayedfor compound concentrations by LC-MS/MS in positive ion mode using aWaters Xevo TQ-S (Waters, Elstree, UK) against matrix matchedcalibration curves and quality control standards. The standards areprepared by spiking aliquots of control plasma, spleen and lung tissuehomogenate with compound and extracted as described for the experimentalsamples. The assay limit of detection 0.168 mg/mL-4000 ng/mL in allmatrices.

Concentrations below the lower limit of quantitation (LLOQ) are treatedas zero for the calculation of mean and SD. Mean concentrations measuredin samples are used to construct semi-logarithmic concentration—timecurve profiles. Pharmacokinetic (PK) analysis is performed usingnon-compartmental methods in Biobook (E-WorkbookIDB5).

Murine Model of Alternaria alternata-Induced Eosinophilic Inflammationof the Lung

Airway eosinophilia is a hallmark of human asthma. Alternaria alternatais a fungal aeroallergen that can exacerbate asthma in humans andinduces eosinophilic inflammation in the lungs of mice (Havaux et al.Clin Exp Immunol. 2005, 139(2):179-88). In mice, it has beendemonstrated that alternaria indirectly activates tissue resident type 2innate lymphoid cells in the lung, which respond to (e.g. IL-2 and IL-7)and release JAK-dependent cytokines (e.g. IL-5 and IL-13) and coordinateeosinophilic inflammation (Bartemes et al. J Immunol. 2012,188(3):1503-13).

Seven- to nine-week old male C₅₇ mice from Taconic are used in thestudy. On the day of study, animals are lightly anesthetized withisoflurane and administered either vehicle or test compound viaoropharyngeal aspiration. Animals are placed in lateral recumbency postdose and monitored for full recovery from anesthesia before beingreturned to their home cage. One hour later, animals are once againbriefly anesthetized and challenged with either vehicle or alternariaextract via oropharyngeal aspiration before being monitored for recoveryfrom anesthesia and returned to their home cage. Forty-eight hours afteralternaria administration, bronchoalveolar lavage fluid (BALF) iscollected and eosinophils are counted in the BALF using the Advia 120Hematology System (Siemens).

Compound activity in the model is evidenced by a decrease in the levelof eosinophils present in the BALF of treated animals at forty-eighthours compared to the vehicle treated, alternaria challenged controlanimals. Data are expressed as percent inhibition of the vehicletreated, alternaria challenged BALF eosinophils response. To calculatepercent inhibition, the number of BALF eosinophils for each condition isconverted to percent of the average vehicle treated, alternariachallenged BALF eosinophils and subtracted from one-hundred percent.

What is claimed is:
 1. A dry powder formulation for inhalation therapies, comprising: (a) a JAK1 inhibitor; (b) a diluent selected from glucose, lactose and mannitol; and (c) magnesium stearate
 2. The dry powder formulation of claim 1, wherein the diluent is lactose.
 3. The dry powder formulation of claim 2, wherein the JAK1 inhibitor comprises particles having a size from 1-5 microns.
 4. The dry powder formulation of claim 3, wherein the lactose comprises particles having a size from 1-5 microns.
 5. An aerosol-based canister formulation, comprising: (a) a JAK1 inhibitor; (b) Lecithin; (c) trichlorotrifluoromethane; and (d) dichlorodifluoromethane.
 6. The aerosol-based canister formulation of claim 5, wherein: (a) the JAK1 inhibitor comprises 24 mg per canister; (b) the lecithin comprises 1.2 mg per canister; (c) the trichlorofluoromethane comprises 4.025 g per canister; AND (d) the dichlorodifluoromethane comprises 12.15 g/canister.
 7. The dry powder formulation of claim 1, wherein the JAK1 inhibitor is a compound of formula (III)

wherein R² is hydroxy-C₁₋₆ alkyl, or a stereoisomer or a pharmaceutically acceptable salt thereof.
 8. The dry powder formulation of claim 7, wherein R² is 2-hydroxyethyl, 2-hydroxypropyl, 2-hydroxy-1-methyl-ethyl, or 2-hydroxy-1-methyl-propyl, or a stereoisomer or a pharmaceutically acceptable salt thereof.
 9. The dry powder formulation of claim 8, wherein the compound of formula (III) is selected from:

or a stereoisomer or a pharmaceutically acceptable salt thereof.
 10. A method of preventing, treating or lessening the severity of a disease or condition responsive to the inhibition of a Janus kinase activity in a patient, comprising administering to the patient a therapeutically effective amount of the dry powder formulation of claim
 1. 11. The method of claim 10, wherein the disease or condition is cancer, stroke, diabetes, hepatomegaly, cardiovascular disease, multiple sclerosis, Alzheimer's disease, cystic fibrosis, viral disease, autoimmune diseases, atherosclerosis, restenosis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, asthma, allergic disorders, inflammation, neurological disorders, a hormone-related disease, conditions associated with organ transplantation (e.g., transplant rejection), immunodeficiency disorders, destructive bone disorders, proliferative disorders, infectious diseases, conditions associated with cell death, thrombin-induced platelet aggregation, liver disease, pathologic immune conditions involving T cell activation, CNS disorders or a myeloproliferative disorder. 